Method for producing purified hematinic iron-saccharidic complex and product produced

ABSTRACT

A method for separating and purifying the active hematinic species (AHS) present in iron-saccharidic compositions, including AHS such as sodium ferric gluconate complex, ferric hydroxide-sucrose complex and ferric saccharate complex and others of similar form and function. The method separates the AHS from one or more excipients and, preferably, lyophilizes the separated AHS. Separation of the AHS permits its analytical quantification, further concentration, purification and/or lyophilization as well as preparation of new and useful products and pharmaceutical compositions, including those useful for the treatment of humans and animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/600,173filed on Jun. 20, 2003, now U.S. Pat. No. 6,929,954, issued on Aug. 16,2005, which is a continuation-in-part of U.S. application Ser. No.10/371,783 filed on Feb. 21, 2003, now U.S. Pat. No. 6,773,924, issuedon Aug. 10, 2004, which is a division of U.S. application Ser. No.09/999,394 filed on Oct. 31, 2001, now U.S. Pat. No. 6,537,820, issuedon Mar. 25, 2003, further claiming the benefit of U.S. ProvisionalPatent Application No. 60/245,269, filed Nov. 2, 2000, the disclosuresof which are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention relates to therapeutically active iron-containingspecies including parenteral hematinic pharmaceuticals. For purposes ofthe present invention a “hematinic” means a compound or compositioncomprising iron in a form that tends to increase the amount ofhemoglobin in the blood of a mammal, particularly in a human. While suchcompounds can be broadly characterized as iron-carbohydrate complexes,which can include dextrans, the present invention is directed to thegeneric subclass known as iron-saccharidic complexes and includes suchspecies as sodium ferric gluconate complex in sucrose (SFGCS), ferrichydroxide-sucrose complex (FHSC) and/or others characterized as ironsaccharates. For purposes of the present invention, such activeiron-containing species are referred to generically as iron-saccharidiccomplexes or active hematinic species (AHS). The term “complex” may havealternate meanings in various contexts in the related art. In oneaspect, the term complex may be used to describe the association betweentwo or more ions to form a relatively low molecular weight non-polymericcomposition which exists singly under a given set of conditions. Thistype of complex has been referred to as a “primary complex”. Analternate manner in which this term is used is to describe anassociation or agglomeration of a plurality of primary complexes into alarge macromolecule, or “secondary complex.” For purposes of the presentinvention, the latter agglomerates are also referred to herein asmacromolecules. For the purposes of the present invention, suchmacromolecules or secondary complexes are identified as “complexes” andare referred to simply as complexes. As an example of the abovedistinction, ferrous gluconate is a composition comprising divalent ironions and gluconate anions. A divalent iron ion and two gluconate anionsform a primary complex of relatively low molecular weight (about 450Daltons) and primary complexes of this type do not become agglomeratedinto macromolecules when dissolved into an aqueous medium. Ferrousgluconate, therefore, is a not composition which falls within the scopeof the term “complex” herein. Ferric gluconate, however, does exist as acomplex as that term is used herein because primary complexes oftrivalent iron ions and gluconate anions agglomerate to form largemacromolecules (and can have molecular weights of from about 100,000 toabout 600,000 Daltons, or more). Several embodiments of therapeuticallyactive ferric iron compounds are commercially available, as will bedescribed below. For purposes of the present invention, the term“excipients” means non-hematinically active components, includingsynthesis reaction by-products and unreacted starting materials,degradation by-products, diluents, etc., present in admixture withtherapeutically active iron-containing species such as iron-saccharidiccomplexes. Such excipients can include one of more sugar, such assucrose, that may be present in combination with the AHS followingsynthesis, as an unreacted or partially reacted component, or added tothe AHS in the course of preparing a parenteral composition, e.g.,commercially available parenteral iron compositions as described below.

Iron deficiency anemia is a blood disorder that can be treated usingvarious therapeutic preparations containing iron. These preparationsinclude simple iron salts such as ferrous sulfate, ferrous gluconate,ferrous fumarate, ferrous orotate and others. Various low molecularweight iron, Fe(III), compounds intended for use as oral or nutritionalsupplements are known. Such low molecular weight compounds are onlyuseful as oral supplements, since the introduction of materials havinghigh unit concentrations of iron directly into the bloodstream byinjection would be contraindicated and could be toxic. In contrast, thecompounds of the present invention, intended for parenteral use, havelower iron concentrations and can be used parenterally. For purposes ofthe present invention, parenteral means introduced into the body by someother means than through the gastrointestinal tract; for example, byintradermal, subcutaneous, intramuscular, intravenous, intramedullary,intra-articular, intrasynovial, intraspinal, intrathecal or intracardiacinjection or infusion.

If the use of such orally administered substances fails to ameliorateiron deficiency, the next level of treatment includes parenteral ironadministration. Depending on a patient's clinical status, parenteraladministration of polyglucan or dextran-linked iron may serve as aneffective therapeutic iron-delivery vehicle. Intramuscular injection orintravenous routes may be used to administer these iron dextrans;commercial examples of such products include those having trade namessuch as “Imferon”, and “INFeD”. Various clinical conditions that requireparenteral iron have shown the practical hematinic value of irondextrans. The use of iron dextrans is tempered by idiosyncrasies intheir synthesis, manufacturing and patient responses such ashypersensitivity. These effects may be exhibited as a severe allergicresponse evident as anaphylaxis or symptoms as minor as transientitching sensations. Whether such allergic or other adverse effects aredue to individual patient sensitivity to the active ingredient or tobyproducts, impurities or degradation products in the parenteralsolution has not been established.

As an alternative to iron dextrans, iron-saccharidic complexes areregarded herein as non-dextran hematinics. Whereas the iron dextranscomprise polymerized monsaccharidic residues, the iron-saccharidiccomplexes of the present invention are characterized by the substantialabsence of such polymerized monosaccharides. Iron-saccharidic complexesare commercially available, for example, under the tradename Ferrlecit,which is identified as sodium ferric gluconate complex in sucrose(SFGCS). The manufacturer states that the structural formula of theproduct is considered to be [NaFe₂O₃(C₆H₁₁O₇)(C₁₂H₂₂O₁₁)₅]_(n), where nis about 200, and as having an apparent molecular weight of350,000±23,000 Daltons. However, it is noted that, based on thepublished structural formula just recited, the formula weight should besignificantly higher, 417,600 (although, as published, the formula isdifficult to accurately interpret). Furthermore, the commercialhematinic composition comprises 20% sucrose, wt./vol. (195 mg/mL) inwater. The chemical name suggests that therapeutic iron (Fe) in thisform is pharmacologically administered as the oxidized ferric formFe(III) as opposed to the reduced ferrous Fe(II) form. Owing to thecharged oxidation state of Fe(III) it has been suggested that gluconicacid (pentahydroxycaproic acid, C₆H₁₂O₇) also exists in a coordinationcomplex or ligand form in a sucrose solution. For purposes of thepresent invention it is to be understood that the chemistry ofgluconate, whether held in a ligand complex with Fe(III) or not, doesnot exempt it from interactions with other carbohydrates that may bepresent, such as sucrose. Thus, use of the term iron-saccharidic complexwill be understood to indicate the existence of a nonspecific andimprecise structure where ionized gluconic acid (gluconate) and sucrosemolecules are tenuously associated by various bonding interactions t-ogive a molecular scaffolding that incorporates Fe(III). Anothernon-dextran hematinic of the present invention is compositionallydescribed as ferric hydroxide-sucrose complex (FHSC). This parenteralhematinic is commercially available under the tradename “Venofer”. Aswith SFGCS, the descriptive name suggests a form of ferric iron,Fe(III), that is present in a spatial complex with sucrose or somederivative of sucrose. Therefore, non-dextran, iron-saccharidiccomplexes of the present invention include SFGCS, FHSC and mixturesthereof. These iron delivery vehicles include an iron-containingstructural complex that, for purposes of the present invention, isdesignated the active hematinic species (AHS).

For purposes of the present invention, the term AHS is usedinterchangeably with iron-saccharidic complex, saccharidic iron deliveryvehicle, and iron saccharate. The term “saccharate” or “saccharidic” isemployed to generically describe iron atom interactions with anotherindividual molecule or its polymers that display a saccharose groupstructurally identified as—CH(OH)—C(O)—

The simplest occurrence of the saccharose group is where the twoterminal positions in a standard Fischer molecular projection model of amolecule appear as an ald- or a keto-group respectively designated as:(—CH(OH)—CHO) or (—CHO—CH₂OH).

This nomenclature format is also described in Zapsalis, C. and R. A.Beck, 1985, “Food Chemistry and Nutritional Biochemistry,” Chapter 6,John Wiley & Sons, pp. 315-321 (incorporated herein by reference to theextent permitted). Such groups and their first oxidation or reductionproducts occur in molecules recognized as monosaccharides that containcarbon atoms with hydrogen and oxygen in the same ratio as that found inwater. By way of example, the aldose sugar known as glucose would havegluconic acid as a first oxidation product and glucitol, also known assorbitol, as a first reduction product. Both the original monosacchariderepresented by the model of glucose and its possible reaction productsretain evidence of the characteristic saccharide group in an oxidized orreduced form. While these structural variations exist, both remainrecognized as monosaccharides and carbohydrates. In practicalnomenclature, the oxidized version of the saccharose group exhibits acarboxyl group which under the appropriate pH conditions will allow itto ionize according to its unique ionization constant and pK_(a) value.When ionized, the oxidized saccharose group is denoted as a “saccharate”or it can be generically described as a saccharidic acid where theionizable proton remains with the oxidized saccharose group. If theionized carboxyl group of the saccharose group is associated with acation such as sodium, a saccharidic acid salt is formed. For example,oxidation of glucose gives gluconic acid and the sodium salt of thissaccharidic acid is sodium gluconate. Similarly, where a ferrous (FeII)cation is electrostatically associated with the carboxyl group ofgluconic acid, ferrous gluconate results. Monosaccharides that arealdoses commonly undergo oxidation to give their saccharidic acidequivalents or, when ionized, monosaccharate forms may interact withselected cations having valence states of +1 to +3. Glyceraldehyde isthe simplest structure that demonstrates such an ald-group whiledihydroxyacetone serves as a corresponding example of a keto-group.Practical extensions of such structures with six carbon atoms accountfor the descriptive basis of two carbohydrate classifications, one formbeing aldoses and the other ketoses. Aldoses and ketoses arerespectively represented by monosaccharides such as glucose or fructose.With many possible intra- and intermolecular reaction productsoriginating from monosaccharides, including the glucose oxidationproduct known as gluconic acid, efforts to complex iron with saccharatescan produce an AHS. For purposes of the present invention, AHS isconsidered to be a more chemically complex embodiment of hematinic ironthan suggested by the generic descriptor sodium ferric gluconate complexin sucrose (SFGCS) or ferric hydroxide-sucrose complex (FHSC), andtherefore, designations including iron-saccharidic complex orsaccharidic-iron delivery vehicle or saccharidic-iron are usedinterchangeably with AHS. Consequently, intra- and inter-molecularreactions or associations from reactions of monosaccharides with ironduring hematinic syntheses can coincidentally produce a wide variety ofstructural species with hematinic properties that are encompassed withinthe present invention.

Typically iron-dextrans are provided for delivery of up to 100 mgFe(III)/2.0 milliliter (mL) of injectable fluid, whereasiron-saccharidic complexes can provide 50-120 mg of Fe(III)/5.0 mLvolume as commercially prepared in a single dose. As made, many of theseiron-saccharidic complex products contain 10-40% weight-to-volumeoccurrences of non-hematinic excipients as well as synthesis reactionby-products.

While some hematinic agents have an established compendial status underthe aegis of the United States Pharmacopoeia (USP) or National Formulary(NF), iron-saccharidic complexes have no acknowledged compendialreference, standardized molecular identity characteristics or documentedmolecular specificity unique to the active hematinic species. Thissuggests that the iron-delivery vehicle in non-dextran hematinics suchas SFGCS or FHSC has not previously been adequately purified andseparated from manufacturing excipients so as to permit detailedcharacterization. Consequently, there has not been developed a benchmarkreference standard or an excipient-free analytical quality control indexcapable of characterizing one desirable hematinic agent from othershaving uncertain characteristics. Since the 1975 merger of the USP withthe NF to produce the USP-NF compendial guidelines for drugs, standardidentities and analytical protocols have been developed for over 3,800pharmaceuticals while 35% of marketed pharmaceuticals are still notincluded in the USP-NF. Hematinic pharmaceuticals such as SFGCS and FHSCfall within this latter category. This issue has been recently addressedin “Raising the Bar for Quality Drugs”, pp. 26-31, Chemical andEngineering News, American Chemical Society, Mar. 19, 2001. As in thecase of immune and anaphylactic responses elicited by specific antigens,a fine line of molecular specificity and compositional differentiationcan separate a no-adverse-effect level for one hematinic's activemolecular structure and excipients from another that may induce suchadverse reactions. Thus, there is a need to identify features thatdocument one hematinic's safe and effective characteristics from otherswhere little is known about the iron-delivery vehicle, excipientsrepresenting synthesis reagent overage or byproducts of hematinicsynthesis reactions. Furthermore, there are no long-term detailed samplearchives or data using modern analytical instrumentation thatmeaningfully characterize the chemical nature of even the safestparenteral iron-saccharidic complexes. Moreover, correlation betweenvariations in normal hematinic manufacturing conditions and theirconsequential effects identifiable as changes in the chemical structureof a released pharmacological agent have not been identified. Themethods of the present invention can address such issues.

The present invention can also provide an analytical basis for a routineprotocol in order to fingerprint and characterize iron-saccharidiccomplexes such as SFGCS, FHSC and others as well as discriminate betweencompeting products and structural transformations exhibited by anindividual product.

The need to characterize an AHS is also reflected in the quality controldemands of manufacturing processes, particularly where endothermicconditions and heat transfer issues can affect final product quality.Whatever the proprietary synthesis process, possible heat-driven orStrecker reaction byproducts in some commercially released non-dextranproducts suggest that hematinic product formation is contingent on atleast some controlled heat-input during the course of manufacturing.Such excipients would not occur if process temperatures less than about50° C. were unnecessary. It follows then, that product quality isrelated, to some extent, to issues of heat transfer rates and durationof heat exposure. Where products are especially sensitive to heatprocessing conditions, knowledge of excipient profiles can also providesignificant insight to the product quality of the active pharmacologicalsubstance. In other words, monitoring the safe and effectivepharmacological agent can also be indicated by the nature and constancyof excipient occurrence in a drug as released into the marketplace.

Analytical studies on iron-saccharidic complexes, including AHS and itscoexisting excipients are hampered by factors of low concentration,molecular interactions, over-lapping analytical signals and so on. Forboth SFGCS and FHSC, analytical challenges include high concentrationsof hydrophilic excipients, including excess reactants and reaction andpost-reaction byproducts, from which their respective AHS's have notpreviously been isolated or reported in terms of their individualproperties. Reference standards for pharmaceuticals need to abide bypractical protocols that are routinely achievable using methods that areanalytically discriminating and able to be verified and validated. Thereis a continuing need for such methods and application of the presentinvention, can facilitate compliance with such protocols as well asverifying manufacturing consistency and product stability.

SUMMARY OF THE INVENTION

A method for measuring at least one molecular characteristic of aniron-saccharidic complex, the complex comprising at least one activehematinic species (AHS) and one or more excipients, the AHS having adetermined value of differential refractive index increment (dn/dc), themethod comprising: (A) subjecting the complex to liquid chromatographicanalysis (LCA) having a refractive index (RI) detector and in-lineeluate stream detection using laser light scattering (LLS); and (B)calculating the molecular characteristics based on the dn/dc value. In aparticularly preferred method the dn/dc value is determined by (a)substantially separating the AHS from the one or more excipients toobtain purified AHS; and (b) measuring the dn/dc value of the purifiedAHS. The method is particularly useful for calculating molecularcharacteristics such as absolute molecular weight, molecular weightdistribution, size, shape, morphology, and dimensional variations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chromatographic signature obtained for isolated andpurified AHS or primary reference standard of an iron-saccharidiccomplex as isolated in Fraction 1.

FIG. 2 shows the chromatographic signature obtained for four isolatedexcipients in Fraction 2, including added trace amount of AHS or primaryreference standard.

FIG. 3 shows refractive index (RI) and laser light scattering (LLS)analytical results for a sample of iron-saccharidic complex partitionedinto Fraction 1 (AHS) and Fraction 2 (excipients) using high pressureliquid chromatography (HPLC).

FIG. 4 shows LLS and RI data based on HPLC analysis of a commercialsample of iron-saccharidic complex and indicating structural deviationsfrom AHS or primary reference standard indicated as active hematinicspecies aggregate peak (AHSAP)

FIG. 5 shows LLS and RI based on HPLC analysis of a second commercialsample of iron-saccharidic complex and indicating structural deviationsfrom AHS or primary reference standard indicated as active hematinicspecies aggregate peak (AHSAP)

FIG. 6 shows LLS and RI data based on HPLC analysis of a sample ofiron-saccharidic complex and indicating an iron aggregate peak(AHSAP_(TAM1)) by time interval 1 after its manufacture.

FIG. 7 shows LLS and RI data based on HPLC analysis of a sample ofiron-saccharidic complex and indicating an iron aggregate peak(AHSAP_(TAM2)) by time interval 2 after its manufacture.

FIG. 8 shows LLS and RI data based on HPLC analysis of a sample ofiron-saccharidic complex and indicating an iron aggregate peak(AHSAP_(TAM3)) by time interval 3 after its manufacture.

FIG. 9 shows LLS and RI data based on HPLC analysis of a sample ofiron-saccharidic complex and indicating an iron aggregate peak(AHSAP_(TAM4)) by time interval 4 after its manufacture.

FIG. 10 shows the chromatographic signature for an iron-saccharidiccomplex isolated as Fraction 1, lyophilized, reconstituted and analyzedusing RI and LLS-based HPLC.

DETAILED DESCRIPTION

Prior to the present disclosure, the AHS responsible for parenteraliron-delivery had not been suitably separated from its excipients withcontrolled, reproducible purity. Such co-mingling of excipients with theAHS challenged the development of improved hematinic products as well ascharacterization of the AHS. In view of the improved methods disclosedherein, opportunities are afforded for analytical monitoring of theiron-saccharidic complex to gauge product compliance with manufacturingspecifications, to determine storage stability indices andbatch-to-batch comparison using a benchmark reference standard. Such abenchmark reference standard has not previously been available,particularly since the AHS had not been isolated. The methods of thepresent invention allow therapeutically active AHS to be effectivelyseparated and concentrated from coexisting excipients, and then to bedried, or lyophilized, if desired. The present invention provides theinformation, methods and analytical details for the preparation,characterization and use of sodium ferric gluconate complex in sucrose,SFGCS, ferric hydroxide sucrose complex, FHSC and iron saccharates. Thematerials are generally referred to as iron-saccharidic complexes orhematinics. It also provides methods for establishing referencestandards for these materials that were heretofore non-existent. Thedisclosure also provides the analytical basis for routine discriminationin connection with manufacturing and continued monitoring of theseproducts after release. The analytical and other methods disclosedherein are generally applicable to iron-saccharidic complexes includingcommercial and generic iron-saccharidic complexes as well as currentcommercial parenteral iron-saccharidic complexes that perform ahematinic function.

Prior to development of the methods disclosed herein, standards foriron-saccharidic complexes were not available because there was nomethod to separate, purify and characterize the AHS without undulyaffecting or destroying the AHS. However, nondestructivecharacterization of the AHS using high pressure liquid chromatography incombination with laser light scattering combine to provide an analyticalmethod for safely and accurately characterizing such saccharidic-ironcomplexes. Coupled with the separation and purification process taughtherein, there is now a method for providing AHS reference standards withthe further opportunity to use such materials in specifying products.

According to the present invention, a method for characterizing the AHSin the absence of its excipients includes separating the AHS andassociated excipients into at least two fractions. Since the AHSordinarily exists at very low concentrations, once separated, it is alsopreferable to concentrate the AHS in order to permit its detailedanalytical study. Although AHS are subject to degradation, they can beconcentrated, provided that hydrophilic excipients are substantiallyabsent from the composition prior to or at the time of concentration.Concentration can be effected by drying and, preferably, bylyophilization, but other methods can be employed. After drying,preferably by lyophilization, the AHS is in powder form.

Weak electron bonding interactions such as hydrogen bonding betweenwater and excipients provide challenges in the separation and drying ofthe AHS. As released, for example in parenteral form, the AHS typicallyis present in an aqueous system that has a lower water activity (A_(w))than solute-free water, i.e., pure, water. It is generally accepted thatsolute-free water has an A_(w) value approaching or equal to 1.0. Wateractivity is a parameter corresponding to the availability of the waterpresent in a substance or composition for participating in physical,chemical and biological processes. Water activity is reduced as aconsequence of the presence of water soluble or hydrophilic substances,in particular those having a low molecular weight. In other words, aportion of the water present corresponding to the fraction of the waterwhich serves to bond or dissolve the substances is otherwiseunavailable. Consequently, water activity diminishes as the numberand/or concentration of substances dissolved or bound increases. As mostoften applied in the food and pharmaceutical industries, elevated wateractivity values are associated with chemical and biological degradationso that water soluble or hydrophilic substances are added in order todecrease the value of A_(w). In the present invention, solutes and/orcolloids, including materials suspended or dispersed in the aqueouscomposition, including saccharidic excipients accompanying the AHS,interact with water, e.g., they are hydrophilic or they can hydrogenbond. Such substances reduce the A_(w) value for water present to avalue less than 1.0. So long as hydrophilic excipients are present inwater in which the AHS is present, a portion of the water remains boundto such excipients and removal of water from the composition comprisingthe AHS, accompanied by its concentration, cannot acceptably beachieved. Consequently, separation of hydrophilic excipients from theAHS increases the A_(w) value toward 1.0 in the phase or fractioncomprising the AHS, and facilitates the concentration, drying andanalysis of these preferred species.

Solute effects on water in systems comprising SFGCS and FHSC directlyimpact upon the drying behavior of these species, includinglyophilization, because lowering of their released hydrophilic andexcipient concentrations is accompanied by an increased water vaporpressure and an associated increase in the A_(w). The concept of wateractivity, expressed as A_(w), has found its most significant use in thearea of food science (see, for example, “Principles of Food Science”,edited by O. R. Fennema, “Part II, Physical Principles of FoodPreservation”, M. Karel, et al., pp. 237-263, Marcel Dekker, Inc. 1975;Enclyclopedia of Food Science, edited by M. S. Peterson, et al., “WaterActivity in Relation to Food”, D. H. Chou, pp. 852-857, Avi Publ. Co.,Inc., 1978, each incorporated herein to the extent permitted). Thepremise is that there is a relationship between the chemical andphysical processes that may occur in food storage and the amount andstate of water in the foods. The same principles can be applied to othersubstances or compositions in which water is present and which can beaffected by water. Water present in a composition, as a solvent, diluentor otherwise, can be characterized as free or unbound or bound tovarious degrees to other compounds, for example, in the present case toexcipients or the AHS. Various methods can be used to determine thedegree to which the water is bound, including determining the amount ofunfrozen water in a water containing composition below 0° C.,measurement by nuclear magnetic resonance, determination of dielectricproperties and measurement of vapor pressure; the latter technique ispreferred for its simplicity. In this method, water activity is definedas the ratio of partial pressure of water in the composition or compoundin which water is present, to the vapor pressure of pure water at agiven temperature. This follows from Raoult's Law where water vaporpressure present in a solution (P) is compared to the vapor pressure ofpure water (solute-free) (P_(o)). As the ratio P/P_(o) increasinglydrops below 1.0, the A_(w) value becomes smaller, the entropy state forsuch water decreases, its vapor pressure is lowered, presumably as aconsequence of increased water-solute binding interactions, and vaporphase removal of water becomes more difficult. These conditionsinfluence drying processes such as lyophilization. Furthermore, incomplex mixtures, in other words, where chemical compounds or complexesmay not be completely dissolved or present in the form of a truesolution, there can be substantial deviations from the ideal Raoult'sLaw expression and, furthermore, water activity of the componentscomprising the composition can differ. It has also been observed inmultiphasic compositions that transport or diffusion of water from acomponent in which Aw is higher to one where it is lower, can occurcontrary to what might otherwise be expected based on concentrationdriving forces (M. Karel et al., page 251). It should also beappreciated that, as a complex composition is dried, the concentrationof solutes having differing degrees of interaction with water, forexample, different levels of hydrogen bonding, can affect, in a complexand changing way, the ability to remove the water. This is a furtherimportant reason to substantially separate or isolate the activehematinic species from their excipients so that appropriate dryingprocess conditions can be determined and controlled. Water can bestrongly bound to specific sites on compounds, including the hydroxylgroups of polysaccharides, as well as to carbonyl and amino groups andothers on which water can be held by hydrogen bonding, by ion-dipolebonds, or by other strong interactions. Therefore, in the present case,water can be bound to specific sites on the AHS and excipients, forexample, as a monolayer. Such water can be present as non-freezablewater, unless temperatures are significantly below 0° C.; additionally,it can be present as non-solvent water. In the present compositions,water is known to bind strongly to sucrose so that A_(w) in asucrose-containing composition is depressed. Therefore, drying,particularly freeze drying (described in detail below), can be adverselyaffected.

Whether lyophilized (freeze-dried) or not, the ability to preparepurified AHS as a discrete substance provides significant capabilitiesfor defining the chemical and structural features that characterize suchhematinic products. With verifiable and reproducible parametersestablished for such a hematinic, it is possible to establish a qualitycontrol framework of, for example, batch-to-batch uniformity of the AHS.Departures from validated structural uniformity in the AHS can be usedas an index of storage stability and to assist in identifyingcause-effect relationships of variables in manufacturing processes. Theability to isolate and analytically characterize these agents usingmodern technology, particularly laser light scattering, helps to definethe attributes of such iron-saccharidic complexes.

Little is known about the synthesis of iron-saccharidic complexes forhematinic use because such syntheses have generally relied onproprietary methods. Similarly, there is little analytical informationavailable for such complexes. The active pharmacological hematinicspecies or iron-delivery vehicle in the hematinic is believed to be lessthan five percent according to weight-by-volume measurements of totalsolids and it may be as low as 1.0 percent. Thus, in commercialproducts, measurement of the characteristics of the AHS as released hastypically been carried out in the presence of a high percentage ofhydrophilic excipients. Such low concentrations of the unique ferriciron-delivery vehicle accompanied by relatively large amounts ofexcipients challenged characterization of the AHS. Furthermore, noreference standard had been established for AHS since it was believed tobe so unstable that it could not be isolated for analysis. With theeffective separation of AHS from its excipients based on the methodsdescribed herein, these methods can be extended so as to developimproved hematinic agents alone or admixed with other pharmacologicalagents. For example, purified AHS can be combined with erythropoietin orother useful drugs.

Access to excipient-free AHS permits closer analytical monitoring of theAHS and its pharmaceutical performance. Moreover, for pharmaceuticalswhere excipients account for a high percentage of the total solidsweight, the ability to independently monitor such excipients issimilarly significant for issues such as quality control, manufacturingand clinical performance. Unanticipated clinical effects can be aconsequence of previously unnoticed or changed distributions and typesof excipients that occur during manufacturing. For food systems, thedevelopment of byproduct compounds due to thermal effects on saccharidesprovides important insights into the adequacy and control of processmanufacturing steps. Such issues have been addressed for other productsby H.-J. Kim, U.S. Pat. No. 5,254,474 while others have focused onthermally generated aldehydes in biotechnology applications, see C. M.Smales, D. S. Pepper, and D. C. James, 2000, “Mechanisms of proteinmodification during model antiviral heat-treatment bioprocessing ofbeta-lactoglobulin variant A in the presence of sucrose,” Biotechnol.Appl. Biochem., October, 32 (Pt. 2) 109-119. Detection of compoundslinked to thermal effects on saccharides in pharmaceutical manufacturingcan also be significant where heat is a factor in synthesis. Indeed,identification of nominal excipient levels and distributions incomplexes such as FHSC and SFGCS can verify that proper synthesisconditions have been employed.

The present invention provides methods for separating hematinicsincluding SFGCS, FHSC and iron saccharates into at least two distinctfractions. Furthermore, the methods are generally applicable to productsin which iron is supplied as an iron-saccharidic complex or an irondelivery vehicle where iron is held in association with negativelycharged carboxyl groups derived from carbohydrates. Initially, onefraction of the hematinic, identified herein as Fraction 1, comprisesthe iron-saccharidic complex or iron-delivery active hematinic species,AHS. Another fraction, identified herein as Fraction 2, comprises amixture of substantially all of the excipients that coexist with the AHSin the original composition, e.g., as synthesized or as released.Excipients in Fraction 2 can be, for example, hydrophilic organic orionic compounds.

The solutes or suspended or dispersed components present in liquidFractions 1 or 2 can be used for detailed analytical study orcharacterization purposes, and can be further concentrated or used asstarting materials in the development of new products. The presentinvention discloses the separate, but related, analytical roles thatthese fractions can play so as to facilitate the establishment ofbenchmark reference standards that characterize sodium ferric gluconatecomplex in sucrose, SFGCS, and ferric hydroxide sucrose complex, FHSC,from their excipients. Furthermore, the present invention disclosesmethods for preparing concentrated, lyophilized and optionallyreconstituted AHS. Separation of the various components is achievedstarting with the as-synthesized or commercially available hematinicwith its combined AHS and excipients in an aqueous (water) compositionand then selectively relocating or extracting the excipients so thatthey are present in a substantially separate aqueous phase. Afterestablishing these two fractions, each can undergo detailed analyticalcharacterization, further purification if desired, and concentration tosatisfy analytical, synthesis or manufacturing goals.

Hematinics are a class of pharmaceuticals designed to conveyhematopoietically useful iron and Fraction 1 comprises from at leastabout 75 to about less than about 100%; preferably; from at least about80 wt. % to about 99.9 wt %; more preferably from at least about 90 wt.% to about 99.9 wt. %; most preferably from at least about 95 wt. % toabout 99.9 wt. %; of the AHS intended for parenteral delivery, whereasFraction 2 comprises correspondingly low levels of the AHS, for example,less than about 0.1 wt. % and substantially all of the excipientsoriginally present in the hematinic composition.

1. Substantially Separating the AHS from Coexisting Excipients.

At least one active hematinic species, AHS, can be isolated orsubstantially separated from excipients present in an as-synthesizedhematinic or in commercially available pharmaceuticals characterized asiron-saccharidic complexes. Such separation has been achieved by theinventors herein as a consequence of their determining severalsignificant characteristics of hematinic compositions which comprise aniron-saccharidic complex, including: (1) that it is necessary toincrease the A_(w) value of the AHS-containing phase or fraction; (2)AHS display at least one detectable iron species with a formula weightof from about 250,000 to about 3,000,000 Daltons or more; (3)manufacturing and stability variations can result in more than onedetectable iron-containing species being present; (4) Fe(III)-containingspecies can display different shapes, e.g., as measured by laser lightscattering; (5) Fe(III)-containing AHS have an electrical charge; and(6) Fe(III)-containing AHS can have a detectable oxidation-reduction(redox) potential indicative of the presence of ferric iron, (Fe(III),or ferrous iron (Fe(II). Separation of AHS from its excipients into atleast two fractions may be achieved by using one or more of thefollowing methods, with or without preliminary pH stabilization in therange of about 6.0 to about 8.0; preferably about 6.4 to about 7.8, morepreferably about 6.6 to about 7.6 for example about 6.8 to about 7.4:

1. Electrokinetic migration where AHS concentration depends on directelectrical current flow that causes electrically charged AHS to depositon a charged collection surface or within an aqueous volume in which acharged surface is present separate from its excipients.

2. Electrokinetic-based membrane technology wherein a cathode and ananode are placed in a water system separated by a semipermeable membranepartition. A direct current applied over the membrane causes theelectrically charged AHS to concentrate on the appropriate electrode dueto attraction between opposing charges. Such AHS concentration on asingle electrode in one compartment allows the dialysis-mediated removalof hydrophilic excipient carbohydrates through the semipermeablemembrane into the accompanying compartment. Preferred semipermeablemembranes include cellulose, cellulose acetate, cellulose ester orregenerated cellulose. In order to maintain retention of the AHS in onecompartment the membrane has a preferred molecular weight exclusion sizeof about 90,000 to about 300,000; preferably about 150,000 to about200,000. Preferred conditions for the method include the use ofdistilled, deionized water at a pH of about 7.5 to about 9.8; a pressureof about 1.0 atm; and a temperature of about 2° C. to about 50° C. Therate of dialysis removal of hydrophilic excipients can be improved byfrequent changes in aqueous dialyzing fluid. The process is generallydescribed by Ficks Law, F=−DA(dc/dx), where F=the total flux; D is thediffusivity of the species in the medium, e.g., water; A is the surfacearea available for diffusion and dc/dx is the concentration gradient ofthe excipients through the membrane.

3. Capillary electrophoresis technology that concentrates AHS fromcoexisting excipients. Capillary electrophoresis, sometimes referred toas capillary zone electrophoresis, relies on the introduction of anelectrically charged analyte within a fused silica capillary with sizedimensions of about 50 to about 75 microns in diameter by about 50 toabout 100 cm in length to which is applied a voltage of up to about 30kilovolts. The differential electrokinetic migrations of chargedsubstances in the composition can be detected and recorded by a varietyof methods described herein, including UV-VIS, fluorescence and massspectroscopy. The final migration positions of the electrically chargedsubstances in the capillary can be documented as an electropherogram.This method can be particularly useful owing to the substantialelectrical charge on the AHS within iron-saccharidic complexes.

4. Column chromatography, which is a particularly preferred process forselective separation of AHS. Owing to the discrete formula weight, sizedimensions, shape and charge of the AHS in contrast to coexistingexcipients, AHS engage in different stationary phase interactions as aliquid carrier or eluent (e.g., water) transports them over and/orthrough a solid support. Thus, differential diffusion or migration ratesresponsible for such separations reflect relative electrical chargeand/or size exclusion differences that retard or accelerate elution ofthese substances through the chromatography system. Separations can alsobe adjusted by modifying the porosity, electrical charge or adsorptionproperties on the surface of the stationary phase. Such chromatographicprocesses may be carried out at from about 3° C. to about 150° C.;preferably from about 15° C. to about 35° C.; typically, 25° C. usingaqueous or non-aqueous solvents supplied to columns in a singular ormultiple serial flow scheme. Chromatographic partitioning of AHS may becarried out at any pressure drop over the inflow and outflow of acolumn. The internal pressure of the column may range from belowatmospheric pressure to any pressure that the column and stationaryphase material can tolerate. Operational pressures above about oneatmosphere (0.1 MPa) are preferred, but pressures up to about 10,000pounds per square inch (69 MPa) may be employed. Eluants supplied to thecolumn can include any solvent or diluent so long as AHS is maintainedas an iron-saccharidic complex. Such eluants include C₁-C₆ alkanols,ethanolamine, dimethylsulfoxide, carbonyl-based solvents,dimethylforamide, water, aqueous buffer solutions and various admixturesincluding water-saccharide solutions. The use of from about 2.0 to about25 weight percent of a primary alcohol may be useful for controllingpotential microbial growth. Suitable stationary phase materials arecommercially available including porous silica, crosslinked polyglucansidentified as dextrans, crosslinked methacrylate polymers, copolymers ofethylene glycol and methacrylate, crosslinked polystyrene, alumina,agarose gels, cyclodextrins and cationic as well as anionic exchangepackings may be used. Particularly preferred stationary phases forcolumn chromatographic separation of AHS from its excipients in ahematinic composition is crosslinked polyglucan or dextran availablecommercially in various grades as Sephadex G-10, G-15 and G-25(Amersham-Pharmacia Biotech., Piscataway, N.J.); and, a commercialcolumn identified as GMPW_(XL) having a 13 micron particle diameter withpore sizes of from about 100 to about 2000 Angstroms and apolymethylmethacrylate backbone (Tosoh Biosep, Montgomeryville, Pa.).When employing solid stationary phase packing, pore diameter in therange of from about 30 to about 9000 Angstroms is preferred; morepreferably from about 100 to about 8000 Angstroms. The dextranstationary phases are particularly preferred for the bulk separation ofAHS, e.g., using low pressure chromatography; and polymethylmethacrylatebackbone is particularly preferred for analytical characterization ofthe AHS, e.g., using HPLC.

The methods of the present invention can be used singularly or incombination with each other in order to separate AHS that is primarilyor substantially responsible for the desired pharmacological actionattributed to FHSC or SFGCS. The substantially separated and,consequently, purified AHS serves as the basis for establishing aprimary reference standard. In turn, the primary reference standard canthen provide a standard for use in monitoring manufacturing andpharmaceutical quality control. If desired, the AHS can be subjected tofurther purification using the methods taught herein. For example, whereanalysis of a hematinic composition using HPLC in combination with, forexample, LLS and RI, shows the presence of a shoulder on, or a peakpreceding the AHS primary reference standard, a further separationmethod can be employed. As described herein, such a shoulder orsecondary peak may be present in a hematinic composition as aconsequence of departure from preferred manufacturing conditions or as aresult of storage of the hematinic composition, particularly in thepresence of hydrophilic excipients. The further separation orpurification process comprises additionally separating the first toelute material from the preparative chromatographic column using theHPLC analytical results or data obtained directly from an LLS detectorin combination with the preparative chromatographic column. In thismanner, a significant percentage or substantially all of the undesirableaggregated AHS can be separated from the preferred AHS. Where an initialnon-AHS peak is present and distinctly separate from the characteristicAHS primary reference standard peak, substantially all of the aggregatedmaterial corresponding to such a peak can be separated from the desiredAHS, for example, in Fraction 1. Where a shoulder appears on the primaryreference standard AHS peak, as will be further discussed below,separation of substantially all of the undesirable, e.g., aggregatedmaterial, may necessarily carry with it some of the preferred AHS or,conversely, not all of the aggregated material may be removed. Theextent of separation and purification can be determined with referenceto the data generated according to the methods of the present invention.Therefore, in addition to removal of substantially all of the lowermolecular weight, primarily hydrophilic excipients present in thehematinic compositions of the present invention, the methods taughtherein can result in an AHS that is substantially purified as well withregard to aggregated iron complexes.

Applying the teachings of the present invention, a discrete activehematinic species (AHS) can be separated from excipients supplied incommercially available parenteral compositions characterized asiron-saccharidic complexes; the separated material can serve as a“primary reference standard.” One method of separating the AHS fromcoexisting excipients employs low pressure gel permeation chromatography(GPC). In the present invention, low pressure refers to operation of thechromatography column at about ambient pressure, including at a pressureslightly above ambient as a consequence of pumping fluid to a packedcolumn to which are attached valved lines and, optionally, otherequipment, including one or more analytical instruments or detectors.This technique can be used not only for analysis of the separatedmaterials, but also for producing bulk quantities of AHS for preparationof new parenteral compositions. Preferably, the column packing comprisesepichlorohydrin-crosslinked polyglucans with demonstrated molecularweight or size exclusion characteristics greater than about 5,000Daltons; more preferably with size exclusion characteristics greaterthan about 1,500 Daltons are preferred. Suitable equipment is availablefrom Amersham-Pharmacia Biotech, Piscataway, N.J. Furthermore,ion-exchange gels with GPC properties and affinity chromatographycolumns are also suitable.

The separated AHS of the present invention, substantially free ofexcipients, generally has a high absolute molecular weight when measuredby the techniques described herein, e.g., HPLC in combination with LLSand RI. Such high absolute molecular weights are typically greater thanabout 25,000 Daltons and can be greater than about 30,000, 50,000,75,000 or 100,000 to about 3,000,000 Daltons or more; for example,molecular weights of about 200,000 to about 2,500,000; about 250,000 toabout 1,000,000; or about 275,000 to about 850,000. These high molecularweights are to be contrasted with the low molecular weights reported inthe literature for compounds intended for use in tablet form. Such lowmolecular weight compounds are less than about 2200 Daltons, moretypically less than about 1700 Daltons and, in the present invention,are separated from high molecular weight AHS when using, e.g., columnpacking having exclusion characteristics of greater than about 5000Daltons. Consequently, as described herein, separation of high molecularweight AHS from low molecular weight excipients distinguishes the AHS ofthe present invention on the basis of molecular weights, even where suchlow molecular weights are as high as 5000 Daltons.

The AHS is typically present in Fraction 1 and the excipients elutethereafter in Fraction 2. Since material elutes from the column in acontinuous fashion, the reference to fractions is based on that portionof material eluted from the column in which there is presentsubstantially all of the preferred or primary reference AHS that hasbeen substantially separated from its excipients. One method for markingsuch a separation of eluted material is to observe where the LLS signalclosely approaches or returns to the baseline value for the mobile phasefollowing the appearance of the initial peak or peaks indicating thepresence of AHS and/or aggregated AHS (as further discussed below). In apreferred method, a solvent reservoir supplies an aqueous-based diluentor solvent by gravity or metered flow to a chromatography column of anyselected diameter or length provided that the length is at least twicethe diameter. The column can be constructed of glass, stainless steel,polycarbonate or another material that is nonreactive with thecomposition and diluents or solvents employed and able to contain astationary chromatographic support material, also referred to as a bed.The bed typically comprises beads having suitable porosity but otherforms of the bed may be manufactured in situ within the confines of thecolumn, such as a poured-in-place porous polymer. In carrying out theprocess of the present invention, initially an aqueous-based solvent (ordiluent), referred to as the mobile phase, is passed over as well asthrough the interstices of a beaded, porous support bed. As the liquidstream or eluate exits the column, it is conducted, e.g., throughtubing, to one or more detectors that analyze the stream in order todetermine its baseline properties. Detectors may be positioned to dealwith a series flow of eluate from one detector to the next or a splitstream flow that allows parallel, multiple detector monitoring. Suchdetectors target eluate characteristics in terms of real time volumetricflow of the stream. Suitable detectors are employed to measure andrecord such stream properties as pH, electrical conductivity,electrochemical reduction potential, refractive index (RI) and otheruseful analytical properties. UV-VIS absorption (A) and refractive index(RI) are preferred detectors for measuring the baseline properties ofthe mobile phase.

Injection of an iron-saccharidic complex, for example a commerciallyavailable parenteral composition, into the aqueous stream leading intothe top of the packed column bed ensures that various constituents inthe composition will be distributed over and through the porouschromatographic beads. Without wishing to be bound by theory, it isbelieved that separation of the different sized chemical speciescomprising the iron-saccharidic complex-proceeds, for example, by asieving effect and, potentially, with hydrogen bonding interactions.Chemical species larger than the pores are excluded from the pores andthey first exit the column in a relatively small eluate volume (V_(e1)).As the flow of the sample continues through the column, progressivelysmaller molecules can become entrained within the pores and subsequentlyexit the column in a relatively larger eluate volume (V_(e2)). Thus,large species such as AHS within the hematinic elute first (V_(e1)) inFraction 1 and smaller excipient chemical species elute later (V_(e2))in Fraction 2. Such a chromatographic separation produces at least twocomponents or fractions, the AHS and its excipients, when applied to theinitial parenteral volume of hematinics including components such asSFGCS, FHSC. As will be described later, larger sized byproduct ordegradation species can also elute with or prior to the AHS. The AHSseparation is substantially complete so that it is separated fromoriginal coexisting excipients, particularly hydrophilic excipients, andincluding those excipients that may have distinctive fluorescentproperties. Consequently, the AHS is produced in a high A_(w) aqueousfraction, Fraction 1, that is substantially free of excipients andsubstantially all of the excipients are present in a second fraction,Fraction 2, characterized by a low A_(w) condition. Acid hydrogen-basebinding (AH-B) dynamics within the column bed can further increase theeffective elution volume (V_(e2)) for excipients from the column inaddition to their size interactions with the chromatographic bed. Acidhydrogen-base binding interactions are generally discussed by Hodge, J.E. and E. M. Osman, 1976, Chapter 3, in “Food Chemistry,” O. R. FennemaEd., Marcel Dekkar, New York, pp. 92-96; and Zapsalis C. and R. A. Beck,1985, “Food Chemistry and Nutritional Biochemistry,” Chapter 10, JohnWiley & Sons, pp. 588-591 (each incorporated by reference to the extentpermitted). For purposes of the present invention the term“substantially free” when used in reference to the separated AHS beingsubstantially free of excipients means that the fraction containing theAHS includes from greater than 80 wt. %; generally greater than about 85wt. %; preferably greater than about 95 wt. %; more preferably greaterthan about 0.98 wt. %; still more preferably greater than about 99 wt. %and most preferably greater than about 99.9 wt. % up to less than orequal to about 100 wt. % of the AHS eluted from the column (in otherwords, there can be present trace amounts of excipients and the amountof eluted AHS may not include a minor or trace amount of AHS that may beretained in the column, tubing, detectors, or lost during processing).Correspondingly, such excipients as are originally present in thecomposition fed to the chromatographic column, or possibly generatedduring processing, are contained in the subsequent, excipient fraction,Fraction 2, and is equal in amount to the above stated values subtractedfrom a value of 100 wt. %. For example, if the AHS fraction comprisesgreater than about 99.9 wt. % AHS, excipients can be present in anamount less than about 0.1 wt. %. Separation of AHS from non-hematiniccomponents, excipients, also typically separates the AHS from sucrosethat may have been added during the synthesis process or, possibly,post-synthesis. To the extent that sucrose can be readily separated fromthe AHS, it is to be understood that the AHS of the present invention isdistinct therefrom, although a chemical formula or structural diagrammay suggest that sucrose is present. Consequently, with regard to thetwo commercially available hematinics, the separated AHS in one instancecorresponds to sodium ferric gluconate complex and, in the other, toferric hydroxide-sucrose complex, although the commercial products maybe identified as being “in sucrose.” Since sucrose is readily separatedfrom AHS by the methods taught herein, for the purposes of the presentinvention, sucrose is an excipient. However, if desired, sucrose can beadded to a composition that includes the AHS of the present invention ifits presence is considered to serve a useful function, e.g., to modifythe characteristics of a parenteral composition.

The presence of the AHS, compared to the excipients, in thechromatographic eluate stream can be observed using laser lightscattering, as described. Additionally, the elution profile of the AHSand excipients can be detected by using one or more different types ofdetectors whose output signals are simultaneously recorded for theeluate stream. In a preferred method, one detector is employed that issensitive to wavelength (λ) using a UV-VIS absorbance detector andanother is a concentration sensitive refractive index (RI) detector. Thedual, independent output signals from these detectors are processed andseparately recorded as two independent ordinate axes (y-axes) against acommon abscissa (x-axis) expressed in units of cumulative eluate volume(e.g., milliliters) or slices (i) as discussed earlier. This method canidentify where so-called Fraction 1, comprising substantially all of theAHS effectively ends and Fraction 2, comprising substantially all of theexcipients, effectively begins. Experimental evidence demonstrates thatthe fractions are sufficiently separated in time so that the AHS can beobtained substantially free of excipients.

According to the Beer-Lambert Law, light absorbance is related to theconcentration of a specific light-absorbing species. The absorbance (A)of a light absorbing species is expressed as:A=εbc  Equation 5

where c is the concentration of a light absorbing species in moles perliter (M/L); b is the light path in centimeters (cm) through the lightabsorbing species; and, ε is a proportionality constant known as themolar extinction coefficient. This proportionality constant is unique toa light-absorbing species at a given wavelength of light. Thisfundamental law accounts for the fact that light absorbance for a lightabsorbing species at a specific wavelength (λ) converts into aquantitative measure of its molar concentration. Hence, positiveincreases in absorbance at a prescribed wavelength for an analytecorresponds to increases in its molar concentration. For monitoring theAHS of SFGCS or FHSC in a chromatographic eluant stream, 435 nm ispreferred because of high extinction coefficient for that wavelength,i.e. 6.590log₁₀, for iron-saccharidic complexes.

For the preparation of substantially excipient-free AHS usingchromatographic separation, the A_(430nm) value is tabulated againstcolumn eluate volume. This can be transformed into a chromatographicelution profile where A_(430nm) ordinate (y-axis) values and eluatevolumes (x-axis) are plotted against each other. As discussed, thelarger AHS elutes from the chromatographic column before smallerexcipients. So long as a change in absorbance (ΔA) with respect tochanges in eluate volume (ΔV) or ΔA/ΔV consistently shows a positiveratio (+ΔA/ΔV) above a solute-free baseline eluent signal, AHSconcentration increases in eluate volume concentration. For a negativeratio (−ΔA/ΔV) the opposite is true. Where the ΔA/ΔV ratio makes atransition from (+) to (−) the chromatographic elution of the AHS ismaximized as a “chromatographic peak.” From this point onward, as thechromatographic profile continues to display a negative ratio (−ΔA/ΔV)the AHS concentration diminishes in the eluate volume. This is true forAHS concentrations so long as the A_(430nm) continues to asymptoticallydecrease and approach an A_(430nm) value of about 0.0. As thismeasurement signals an end to the elution of the AHS, the concentrationsensitive RI detector begins to respond to increasing amounts ofhydrophilic and other excipients included in the eluate. As above, apositive (+) change in the ratio of RI with respect to changes in volume(+ΔRI/ΔV), signals increasing concentration of excipients. At the eluatecollection volume where ΔA/ΔV still displays a negative ratio (−ΔA/ΔV)asymptotically approaching a value of about 0.0 A and RI-detector beginsto signal a +ΔRI/ΔV slope, this marks the effective boundary whereFraction 1 substantially ends and Fraction 2 effectively begins.Fraction 1 comprises the AHS in a high A_(w) environment and Fraction 2comprises the hydrophilic and other excipients in low A_(w) environment.In this manner, an elution profile is determined.

The eluate boundaries that define the separation where Fraction 1substantially ends and Fraction 2 effectively begins can alternativelybe determined by measuring the percent spectral transmission of the AHSin column eluate at 430 nm. A suitable instrument for this purpose is a“Color Quest XE” system manufactured by Hunter Associates Laboratory,Inc., Reston, Va. Increases in the light transmission of the eluate asthe AHS present in the eluate asymptotically approaches zero percent,can indicate substantial separation or an end to elution of the AHS. Asubsequent increase in RI-response of the eluate stream marks thebeginning of the eluate volume comprising excipients. In a furtheralternative embodiment, a demarcation between where Fraction 1substantially ends and Fraction 2 effectively begins can be determinedby measuring the A_(620nm) anthrone-based absorbance of the eluatestream. Since both the AHS and the excipients have notable dextroseequivalent (DE) absorbency, the detectable A_(620nm) DE-value will beminimal between Fraction 1 and Fraction 2. The principles thatunderscore this analytical concept have been described by R. Dreywood,“Qualitative Test for Carbohydrate Material,” Indus. and Eng. Chem.,Anal. Ed., 18:499 (1946); J. E. Hodge and B. T. Hofreiter,“Determination of Reducing Sugars and Carbohydrates,” MethodsCarbohydrate Chem., 1:384-394 (1962); and more recently by C. Zapsalisand R. A. Beck, “Food Chemistry and Nutritional Biochemistry,” Chapter6, John Wiley & Sons, pp. 353-354 (1985) (each of the disclosures ofwhich are incorporated herein to the extent permitted).

The methods of the present invention allow for preparation of largeproduction batches (for example, from greater than about 500 milligramsto greater than or equal to about 1.0 gram; or from about 1.0 gram toabout 10 grams or more; from about 1.0 gram to about 100 grams or more;from about 1.0 gram to about 1 kg or more; for example, if desired,hundreds or even thousands of kilograms can be produced by the presentmethod) or small analytical samples (from about 5.0 to about 500.0milligrams) of the AHS present in iron-saccharidic complexes of any typecan be prepared. Since the AHS is the preferred component of complexediron present of these parenteral compositions, the ability to separatethe AHS forms the basis for the primary reference standard. Separationof Fraction 1 permits further detailed chemical and/or structuralanalyses; such methods are described elsewhere herein. However isolated,the AHS, e.g. Fraction 1, as well as the excipients, e.g., Fraction 2,may be further concentrated for detailed study.

The present invention is also suitable for production of small scaleamounts of AHS (from less than about 1.0 mg to less than about 5.0 mg),preferably by way of high pressure (or high performance) liquidchromatography (HPLC). An appropriate chromatographic solid support canbe used to separate the AHS from its excipients. This results in AHStransfer into a hydrodynamic volume within the column that displays ahigh A_(w), e.g., approaching a value of 1.0. The excipients aretransferred into a low A_(w) hydrodynamic volume having a low A_(w),e.g., less than the value of the AHS-containing phase. The operationalprinciple for this method is similar to that of the low pressurechromatographic method described above, but the stationary column bedmaterials for the HPLC method are more finely divided so as to withstandpressures of from about 5,000 to about 10,000 pounds per square inch(about 35 to about 69 M Pa). This results in slower flow rates of eluatefrom the column, but it is more than compensated for by the highhydrostatic pressure. A silica-based stationary bed that depends onadsorptive-desorptive analyte separation phenomena can be used toproduce a separation of the AHS from its excipients. Such silica-basedseparation performances however are difficult to control and requireaccurate preparation of an azide-containing aqueous mobile phase;consequently, they are subject to greater analytical errors.Furthermore, shedding of particulates from silica-based beds complicatesor can preclude effective use of a LLS detector. Thus, a polymeric HPLCcolumn, such as a GMPW_(XL) brand manufactured by Tosoh Biosep,Montgomeryville, Pa., that employs an aqueous mobile phase is preferredover silica-based columns. Such HPLC analysis of, for example, acommercially released form of iron-saccharidic complex requirespreliminary 0.02 micron filtration through, for example, an Anotop 25brand inorganic membrane (Whatman, Maidstone, UK).

2. Methods for Characterizing the AHS Primary Reference Standard andCoexisting Excipients.

Analysis for iron reveals that more than about 90% of the iron intendedfor hematinic purposes resides in the high A_(w) fraction designated asFraction 1 above, generally from at least about 75 wt. % to less thanabout 150 weight %; preferably from about 80 wt. % to about 99.9 wt. %;for example, from about 90 wt. % to about 99.9 wt. %; from about 95 wt.% to about 99 wt. %; and excipients reside in the low A_(w) fractiondesignated as Fraction 2. Iron atoms in Fraction 1 can be quantified byatomic absorption spectroscopy (AAS) but iron quantification by AASalone is not determinative of hematinic functionality of the products ofthe present invention.

The relevant characteristic of a non-dextran hematinic of the presentinvention is based on its ability to deliver a physiologically tolerableor benign source of ferric iron, Fe(III), preferably via parenteralmeans. This ferric iron composition is a parenterally acceptable speciesthat resembles properties of an association colloid. An associationcolloid is a reversible chemical combination due to weak chemicalbonding forces wherein up to hundreds of molecules or ions aggregate toform colloidal structures with sizes of from about 1 to about 2000nanometers or larger. Such colloids of ferric ions interacting withsaccharidic compounds exhibit directional migration in an electric fieldin addition to optical activity identified by laser light scattering(LLS) LLS properties relevant herein relate to the Tyndall effect wherean incident light beam (I_(o)) passing through a colloid emerges from itat a 90° angle to its original path. Light scattering only occurs if thelight interacts with macromolecules such as starches, proteins or othercolloidal species where the wavelength of incident light approaches sizedimensions of the molecules. Light scattering can occur as destructiveinterference where the scattered wavelengths interact to cancel eachother out or by constructive interference where two wavelengths of lightreinforce each other. Mathematical evaluation of LLS data permits sizeand shape evaluations of various colloidal species. Size, for example,may be estimated in terms of molecular weight for a single molecule orthe formula weight for a multi-molecular or ionic aggregate. The weightexpressions in either case represent the sum of atomic weights of allatoms present in such structures. The structural diversity of mostaggregates or molecules such as polymers is such that they exist as afrequency distribution of varying weights, typically expressed as anaverage or mean molecular weight distribution (MWD). Apart from size,colloidal shape can have important implications. For example, if itsshape is that of a thin rod, a random coiled structure or a sphere itsinteraction with other molecules or structures can vary. LLS, includingmulti-angle laser light scattering (MALLS) or low angle laser lightscattering (LALLS), combined with one or more methods of HPLC-integrateddetector analysis can be used for evaluating iron-saccharidic complexes.For purposes of the present invention, reference to LLS should beunderstood to include MALLS, the latter being a preferred type ofdetector. The use of LLS measurements herein provides a superior andpreferred analytical method for characterizing an iron-saccharidiccomplex that is normal, in other words, represents the preferred AHSresulting from suitably controlled synthesis, or one that displaysevidence of decay or degradation products. The fundamental mathematicalrelationships and operation of HPLC in combination with laser lightscattering and refractive index detectors for the characterization ofmacromolecular structures and association colloids has been reported.(see P. Wyatt, Light scattering and absolute characterization ofmacromolecules, Analytica Chimica Acta. (1993) 272:1-40; incorporated byreference to the extent permitted). As discussed herein, such techniquesare applicable to iron saccharidic complexes comprising AHS.

Acquisition of LLS data is particularly useful for identifying AHS insaccharidic-iron complexes, particularly where a benchmark analyticalreference standard is required. LLS data can be obtained from anindividual batch based on an isolated sample or LLS data can be obtainedusing in-line analysis of an eluate stream from a continuous processcontaining the AHS following its liquid chromatographic isolation. Inother words, once a saccharidically bound ferric iron complex has beenproduced and examined by LLS alone or in combination with other methodssuch as those disclosed herein, batch-to-batch comparisons ofmanufacturing continuity can be routinely verified in terms of formulaweight as well as morphology of the pharmacological species.Characterization of AHS size and shape features can be used to monitorAHS manufacturing processes, AHS product quality and AHS stability byusing LLS algorithms that are integral with the operational software ofLLS enhanced HPLC. Specifically, as taught herein, size and dimensionalattributes for so-called normal AHS (e.g., primary reference standardmaterial or the preferred AHS) and so-called abnormal AHS (e.g.,degraded or aggregated AHS) structures are determined from a standardDebye plot generated by operating software such as ASTRA (WyattTechnology Corp., Santa Barbara, Calif.). The details of such anapplication described in P. Wyatt, Light scattering and absolutecharacterization of macromolecules. Analytica Chimica Acta, 272:1-40(1993), incorporated herein by reference to the extent permitted.Preferred AHS has been observed to have a generally spherical shape andto be about 10 nanometers or smaller in size, whereas degraded oraggregated AHS, such as material that elutes from a chromatographiccolumn before the preferred AHS, tends to be greater than about 10nanometers in size, for example from about 10 to about 30 nanometers ormore, for example, from about 20 to about 30 nanometers or more. Sizeand shape dimensions can be relevant factors for physiological andmetabolic tolerances as well as for their tissue disposition of ahematinic. Thus, the application of LLS methods is preferred for routinecharacterization (including after manufacturing and during storage) andmanufacturing monitoring for the hematinics of the present invention.

Laser light scattering of AHS provides characterization parameters interms of absolute formula weight or absolute molecular weight. However,since the AHS is not a monomolecular species, but instead behaves as anassociation colloid, the use of absolute molecular weight isparticularly preferred. Such LLS-based molecular weight measurements arepreferred over other methods of weight estimation that provide aso-called relative molecular weight. Relative molecular weightmeasurements of the AHS do not rely on the size and shape dependentphysical interactions of light with an AHS as a basis for estimating itsweight. Instead, relative molecular weight measurements rely on standardmethods of size exclusion chromatography (SEC), also sometimes referredto as gel permeation chromatography (GPC). Both SEC and GPC arehereinafter used interchangeably. In GPC analysis, the effective size ofa macromolecule or aggregate such as an AHS, not its formula weight,determines its elution volume and exit from a calibrated chromatographycolumn. Hence, the relative elution and transit of the AHS through a GPCcolumn relative to a series of calibration standards in a narrow bandabove and below the expected weight of the AHS provides the basis forassigning a relative formula weight to the iron-containing complex.Using this concept, a concentration sensitive detector such as arefractive index (RI) detector would be used for detection of the AHSand its calibration standards as they elute from a column. As aconcentration sensitive detector, RI-measurements rely on the changes inthe indices of light refraction (n) as analyte (for example, solute)concentrations (c) increase or decrease in their respective flow throughthe RI-detector cell. At evenly spaced time intervals, ratios of suchchanges in refraction and concentration are recorded as dn/dc values.Each dn/dc value is unique to a specific analyte or solute and the dn/dcvalue characteristic of one substance is not an a priori valueuniversally applicable to any other specific substance. Such dn/dcvalues recorded for the elution of known calibration standards andunknown analytes can be acquired and recorded at precise time intervals.Each dn/dc measurement in the overall elution profile of a liquidchromatography column is referred to as a slice (i). The slice number,its elution time or elution volume can be used to reference this slice.Elution time of a slice multiplied by the flow rate of the eluent to thecolumn gives the elution volume. Records of slice numbers in terms ofvolume or number do not compromise the general significance of thisdata-recording concept. Thus, a plot of dn/dc (ordinate) versus slicenumbers (abscissa) over the course of a GPC measurement gives an elutionprofile for calibrating standards with respect to any unknown substancesthat may be totally unrelated to each other, yet they havedn/dc-detectable masses suitable for instrumental monitoring.

Because standard GPC procedures do not recognize significant physicalinteractions common to all large molecular or colloidal structures, GPCweight evaluations for the AHS present in SFGCS, FHSC or other similarhematinics, can be susceptible to analytical errors. Furthermore,RI-based GPC methods are unable to characterize the shape andtopological features of the AHS including, for example, structuralbranching. Also, estimations of relative formula weight usingRI-dependent GPC methods are also subject to data variations, aresensitive to minor analyst errors and completely insensitive todimensional variations of species within association colloids that havea high density. For association colloids, and FHSC and SFGCS inparticular, these features are important because nonsaccharidicallyaggregated iron atoms can coexist along with the desirediron-saccharidic complex containing the AHS. Thus, it is important torecognize that RI-based GPC can provide relative formula weight valuesfor analytes of AHS with reasonable accuracy and reasonable precision,but the method overlooks other important characteristics of AHSstructure.

Based on well-known principles of light scattering physics, isotropicparticles with small dimensions such as macromolecules and colloids,interact with light so as to permit the calculation of their absoluteweight and shape. This is true for the AHS contained in FHSC and SFGCS.Unlike GPC-RI based analysis, the absolute formula weight for acolloidal species can be determined independent of any calibration curvethat depends on pre-established graded molecular weight standards (M. J.Vold and R. D. Vold, 1964, “Colloid Chemistry,” Reinhold, N.Y.; andZapsalis, C. and R. A. Beck, 1985, “Food Chemistry and NutritionalBiochemistry,” Chapter 8, John Wiley & Sons, pp. 507-547 hereinafter,Zapsalis and Beck, 1985) (each incorporated by reference to the extentpermitted). It is established that the intensity of light scattered(I_(θ)) by a particle through an angle of θ depends on the intensity ofthe incident light beam (I_(o)), the light path distance (γ_(s)) throughthe light scattering volume and the polarizability (α) of the particle.For unpolarized light the equation isR _(θ)(1+cos 2 θ)=I _(θ)γ_(s) ² /I _(o)=8π⁴/(1+cos 2 θ)  Equation (1)

The term R_(θ) (1+cos 2 θ) is the basis for the Rayleigh ratio.Extension of this light scattering measurement to very dilute solutionsof particles with sizes smaller than the wavelength of incident light(I_(o)) gives the expressionR _(θ)=2π²η_(o) ²[(η−η_(o))/C] ² /Lλ ⁴ ·CM=K*CM  Equation (2)where R_(θ) becomes the Rayleigh ratio (R); η is the refractive index ofa solution containing particulate species, η_(o) is the refractive indexof a solvent without particulate species; C is the concentration ofsolute in terms of mass per unit volume; M is the molecular or formulaweight respectively for a molecule or particulate species; L isAvagadro's number; λ is the wavelength of light; and K*=2π²η_(o)²[(η−η_(o))/C]²/Lλ⁴ is the optical constant. This expression provides abasis for establishing the Rayleigh ratio (R) or that fraction ofincident light (I_(o)) scattered by a particle when a wavelength (λ) ofincident light is comparable to or larger than the size of someparticulate species. Accordingly, R is related to formula weight of ananalyte such as the AHS in SFGCS, FHSC and other iron-saccharidiccomplexes by the physics of light scattering phenomena and not relativecomparisons to GPC calibration curves based on unrelated materials.Independent of instrument detector geometry for detecting the Rayleighratio (R) when incident light (I_(o)) interacts with particulates, arelationship stands whereR=K*CM  Equation (3)

Consistent with in-stream eluate measurement concepts detailed above,where dn/dc values are acquired for GPC-RI based systems, Rayleigh ratio(R) measurements are acquired for each slice in other words “i”, R_(i),and the Raleigh ratio is simply the product of each slice'sconcentration (C_(i)), molecular weight (M_(i)) and the optical constant(K*) or,R _(i) =K*C _(i) M _(i)  Equation (4)

Light scattering also allows for characterization of structuralconsistency of the AHS in iron-saccharidic complexes based ondissymmetry ratios between light scattered at some forward angle θ andthat light scattered at its supplementary angle 180−θ (I_(θ)/I_(180−θ)).Of all possible light scattering angles, about 45° and 135° serve asinstructive reference points. When a plot of I_(θ)/I_(180−θ) (ordinate)versus L/λ (abscissa) is constructed, the structural dissymmetries forspheres, rods and random coils are readily determined by way ofreference to Zapsalis and Beck, 1985, p. 535.

If the AHS is substantially isolated, such as Fraction 1 discussedabove, it may be harvested in (a) bulk production amounts or (b) smallanalytical amounts typical of that found in liquid chromatographiceluate streams. In either case, LLS methods offer a preferred analyticalmethod for establishing a routine reference standard for anyiron-saccharidic complex in this pharmaceutical class.

The ability to produce purified AHS or iron-saccharidic complex usingteachings of this disclosure allows it to be comprehensively analyzedand characterized, including defining its colloidal and molecularattributes. Suitable analytical and characterization methods applicableto the AHS, and to SFGCS and FHSC in particular, include ultraviolet(UV) spectrophotometry, visible (VIS) spectrophotometry and combinedUV-VIS spectrophotometry, including photodiode array (PDA) methods,infrared (IR) spectroscopy, electron spin resonance (ESR), pulsepolarography, energy dispersive X-ray analysis (EDS), circular dichroism(CD) and optical rotatory dispersion (ORD), fluorescent spectroscopy,polarimetry, pyrolysis, and pyrolysis mass spectroscopic analyses,nuclear magnetic resonance spectroscopy, differential scanningcalorimetry (DSC), liquid chromatography integrated with massspectroscopy (LC-GC), matrix assisted laser desorption/ionization massspectrometry (MALDI-MS) techniques, analysis utilizing radioactiveisotopes including radioactive iron, antibodies to hematinic substances,capillary electrophoresis and inductively-coupled plasma spectrometry,atomic absorption analysis, electrochemical analysis, as well asspecific pH storage and stability studies and targetedglucosidic-degradation of the AHS with saccharidic product analysis. InMALDI-MS, laser light is used to ionize a macromolecular analyte anddesorb its ions from a sample matrix into a vacuum system. The method isused to measure the mass of a wide variety of macromolecules inconjunction with mass spectrometry.

Furthermore, the ability to separate the AHS into at least twofractions, Fraction 1 (high A_(w) fraction), and Fraction 2 (low A_(w)fraction), also permits independent analysis of excipients that areconcentrated in Fraction 2. Discriminative excipient fingerprints can beused to characterize the AHS as well as contribute to productidentification and monitoring quality control and assurance. Theoccurrence and verification of typical or expected excipients supportsstandardization and monitoring of manufacturing. This improves thelikelihood for favorable patient use in clinical settings where thehematinic is administered. Since excipients can account for over 75percent of the total solids in typical commercial parenteralcompositions, excipient verification and analysis can be an importantmatter. Moreover, since thermally treated saccharides are known toproduce identifiable chemical markers that reflect their processinghistory, these as well as surplus reactants and byproducts developedduring hematinic manufacturing (for purposes of the present invention,all such materials are generally included in the term “excipients”) canbe used to monitor and characterize both the hematinics and processesused to produce them.

There are at least three possible manifestations wherein ferric ionsinteract or exist in hematinic iron-saccharidic complexes. Firstly, thepreferred principal agent, namely the AHS, behaves as an associationcolloid and it is the desired iron-saccharidic delivery vehicle.Secondly, high formula weight aggregates can develop from the AHS andthey may also be detected. Thirdly, iron can exist as a complex withsurplus saccharidic reactants and/or byproducts of synthesis reactionsteps. This form of iron can be found with hydrophilic excipients inFraction 2. It is particularly preferred that parenteral compositions bemonitored, as well as Fraction (2), for (a) iron content, (b) residualamounts of saccharidic reagents, and (c) evidence of thermally-dependentsynthesis reaction byproducts. A particularly preferred method formonitoring such analytes includes the use of liquid chromatographicanalysis with refractive index (RI) and in-line eluate stream detectionusing at least one of the following: laser light scattering (LLS),electrochemical detection (ECD), photodiode array (PDA) based UV-VISspectrophotometry, infrared (IR) spectroscopy, and liquid chromatographycoupled with mass spectrometry (LC-MS), and, optionally, one or more ofthe analytical and characterization methods described above. Whereas RIis a concentration sensitive detector that allows quantification ofexcipient saccharides, ECD detectors respond to metal andnonmetal-containing compounds having characteristic electrochemicaloxidation-reduction potentials and UV-VIS based PDA analysis permitsdetection of thermally produced saccharidic derivatives in addition tolow formula weight iron-complexed species.

The methods of the present invention further-include methods forcharacterizing the AHS and its coexisting excipients. Application ofHPLC to a previously prepared sample of Fraction 1 comprising AHS usingthe polymeric column described above, in combination with in-stream dualRI and LLS detectors of the HPLC eluate provides the results illustratedin FIG. 1. This figure shows the separated and purified chromatographicsignature for the AHS present in Fraction 1 and, furthermore, that it isfree of excipients. Such an HPLC elution signature is a preferred resultfor a manufactured hematinic comprising an AHS, for example a productreleased in a sealed glass ampoule. FIG. 2 illustrates thechromatographic signature for four excipients present in Fraction 2obtained from the same sample that provided Fraction 1. In this test, asmall amount of the AHS from Fraction 1 was intentionally introducedinto Fraction 2 so that relative positions of the excipients to the AHScould be observed. It is worth noting in FIGS. 2 and 3 that excipientsare better monitored by an RI detector because RI is a concentrationsensitive property whereas an LLS detector is mass sensitive andresponds better to AHS than to excipient carbohydrates. These effectsare clearly shown in FIG. 2 where only a trace amount of pure AHS wasadded as an internal benchmark for relative measurement of excipientelution progress. RI in this case fails to sense any occurrence ofpurified AHS yet the LLS signal records its presence as an analyticalspecies. FIG. 3 further illustrates that the LLS and RI-based HPLCmethod using a polymeric column can be used to analytically separate theAHS from its excipients. That is, the AHS component is separated into ahigh A_(w) hydrodynamic volume, while at the same time the coexistingexcipients that originally accompanied the AHS are substantiallyseparated into hydrodynamic column volumes that elute after elution ofthe AHS. These results are consistent with those in FIG. 1 for theprimary reference standard previously isolated as a discrete entitywhereas FIG. 3 represents results for a multicomponent initialcomposition (AHS and excipients). This confirms that the methods of thepresent invention can be used to monitor hematinic compositions such asthose produced commercially that comprise AHS and excipients.

Conditions such as instability and aging of the AHS and manufacturingvariability can be monitored in iron-saccharidic complexes using HPLCanalysis equipped with at least LLS as one detector mode. Furthermore,LLS-based HPLC used in conjunction with a concentration sensitivedetector, such as RI, can be used to monitor structural variations inthe nominal chromatographic peak signature for an AHS. Such variationscan be seen in the chromatograph in FIG. 4, particularly when comparedto that produced by the primary reference standard in FIG. 1 or theseparated mixture in FIG. 3. Similarly, FIG. 5 shows the presence of analtered AHS peak in another manufacturer's hematinic product. It isnoted that different detectors can provide different information, forexample, the LLS-chromatographic detector and the concentrationsensitive RI-detector. The concentration sensitive RI-detector sensesand records analyte concentration but not its mass, which isindependently documented by the LLS-detector. Thus, the LLS-baseddetector can sense increasing mass of a species formed as a consequenceof, e.g., complexing or cross-linking of AHS, that elutes before the AHSprimary reference standard. Since the AHS, as illustrated in FIG. 1, isthe desired hematinic substance, the species shown in FIGS. 4 and 5,represent even higher formula weight byproducts that are believed toarise from the AHS. The new peak appears to develop at the quantitativeexpense of the preferred AHS.

The methods of the present invention can be used to monitor storagestability of hematinics comprising an AHS. Like most organic molecules,AHS is subject to structural instability, such that it can degrade ortransform, even within sealed glass delivery ampoules used forhematinics, to give new species. Such structural transformations of anAHS are time dependent and may also be promoted by temperature. Evidenceof structural AHS transformation over time is evident for a series ofLLS and RI-based HPLC profiles for iron-saccharidic complexes stored insealed glass delivery ampoules at room temperature in the dark for 6,12, 22 and 25 months after manufacture. The pertinent chromatographicprofiles for these products correspond, respectively, to FIGS. 6, 7, 8and 9. Evidence of structural change is apparent when comparing theunsymmetrical AHS peaks in these figures against the preferred andsymmetrical primary reference standard peak exhibited by the AHS inFIG. 1. The new entity indicative of AHS degradation, and detected byLLS, elutes from the column before the primary reference standard orAHS, which also can be seen in the figures. The new entity has featuresof a very dense, high formula weight structure, conceptually similar to“BB shot”. These characteristics are determined based on the Debye plotgenerated by processing the light scattering data using the ASTRA-brandsoftware incorporated in the LLS equipment, referred to earlier (WyattTechnology Corp., Santa Barbara Calif.), and the methods andcalculations described in the 1993 Analytica Chimica Acta journalreference, also referred to above. Debye plots permit the acquisition ofspecific LLS data which, in conjunction with root mean square (rms) orradius of gyration (R_(g)) measurements, permit size determinations ofmolecular species or colloids. For FIGS. 6-9, the rms-value indicatingparticle diameter sizes gives an average value equal to or greater than20 nm. The corresponding rms-value for the chromatographic signature ofAHS in FIG. 1 is less than or equal to 10 nm; well below the practicaldetection limit of LLS. Thus, the methods of the present invention canbe used for monitoring the quality and storage stability of a hematiniccomposition comprising AHS.

Commercially produced hematinics can be monitored for the AHS, as wellas for excipients, during the manufacturing process, at the conclusionof manufacturing, e.g., at the time of packaging, and after manufacture,e.g., if the product is stored. Similarly, an iron-saccharidic complexcomposition comprising the AHS after separation of the excipients, suchcomposition in the form of an aqueous composition or after drying, e.g.,by freeze drying, can also be monitored in the same manner. Theiron-saccharidic complex comprising the AHS can be monitored not onlyduring manufacturing, but also as it is stored from shortly aftermanufacture, such as from about one week thereafter, as well as after amoderately short storage period of about 6 months to for as long asabout five years or more after manufacture; extended storage can be fromabout 1 year to about five years; or from about 1 year to about 3 years.In each instance, the AHS can be monitored by comparison of itsproperties, including various analytical properties as discussed above,e.g., the chromatographic signature obtained using HPLC in combinationwith LLS and RI to a primary reference standard.

As described above, the smaller LLS-detectable peak resulting fromdegradation or modification of the AHS originally present is anextremely high formula weight substance, identified in FIGS. 6-9 as anAHS aggregate peak (AHSAP). The substance responsible for thischaracteristic AHSAP has a formula weight in the range of from about350,000 to about 3,000,000 Daltons or more based on light scattering,but ultracentrifugation and other methods can also be used to establishthe high formula weight of this species. It is believed that the AHSAPis unrelated to excipients normally occurring as a consequence of theAHS synthesis or manufacturing process. The AHSAP appears, instead, tobe related to an aging phenomenon, but it may also result fromsignificant departures from preferred manufacturing conditions. Themethods of the present invention can be used to identify the levels ofquantitative tolerance of the AHSAP in a parenteral hematinic product.The occurrence and detection of an AHSAP at any time after manufacture(AHSAP_(TAM)) of an iron-saccharidic complex can be compared to thetotal quantitative HPLC signal for the AHSAP detected after a prescribedstorage period of the hematinic while exposed to defined conditions(AHSAP_(TOTAL)). By way of example, the storage period could cover anyconvenient period of time, for example from about 6 months to about 10years or longer; alternatively from about 1 to about 8 years; or fromabout 1 to about 5.0 years. If, for example a 5 year period is used, at5 years the AHSAP_(5yrTOTAL) would provide the basis for establishingthe maximum acceptable decomposition or modification of the measuredcharacteristics of the hematinic after its release. In other words, bythe time 5 years had elapsed the AHS may have decayed intopharmacologically useless iron aggregate and compositional residuesdistinctly unlike the intended embodiment of the initially releasediron-saccharidic complex for parenteral use. Thus, the AHSAP_(TAM)quantified at any time up to AHSAP_(5yrTOTAL), expressed as anoccurrence ratio, [AHSAP_(TAM)]/[AHSAP_(5yrTOTAL)], can provide atime-dependent stability ratio or index for gauging hematinic qualityand actual percent composition of intact AHS remaining in a hematinicproduct. There is currently no such standardized way to quantitativelyaddress aging and decay of iron-saccharidic complexes once they arereleased into commerce. It should be appreciated that the occurrenceratio is not limited to use of the 5 year elapsed time interval, but,rather, as noted above, it applies to any convenient elapsed timeinterval selected. It can be useful to gauge iron-saccharidic complexaging and stability against defined conditions where, for example, 50percent of the iron-saccharidic complex remains in its original form atthe time of manufacture or release into the marketplace, as compared toiron other than iron-saccharidic complex; for example free or unboundiron or iron aggregate, thereby allowing determination of aniron-saccharidic complex half-life. As the [AHSAP_(TAM)]/AHSAP_(TOTAL)]ratio approaches a value of about 0.5, this 0.5 ratio-value can serve asa pharmacokinetic index and guardrail to ensure that the manufacturedproduct will have at least 50% of its iron-saccharidic complex stillintact, as intended for initial release. Although the basis forquantitatively gauging the shelf life of hematinics here cites a valueof 50% survival for iron-complexation in an AHS, practical qualitystandards higher than 50% are more desirable; as a practical matter,from about 0.5 to about 0.98; preferably from about 0.75 to about 0.95;more preferably from about 0.80 to about 0.99; for example, any singlevalue between about 0.5 and less than or equal to about 1.0 (andcorrespondingly, for iron other than iron-saccharidic complex, AHS,values of from 0.02 to less than about 0.5; from about 0.05 to about0.25; and from about 0.01 to about 0.20; for example, any single valuefrom equal to or greater than about 0 to less than or equal to about0.5) may be established by a standards setting organization or by themanufacturer. Such an established ratio of [AHSAP_(TAM)]/AHSAP_(TOTAL)]is particularly useful for purposes of indexing, warranting orstandardizing the clinical efficacy, performance and safety of suchhematinics. Prior to the present invention, there was no basis forassigning quality compliance standards to iron-saccharidic complexesusing standards that are generally applicable to drugs. As describedherein, the use of the HPLC chromatographic method, preferably includingLLS and RI-based detectors allows implementation of such indices.Furthermore, the ability to isolate the AHS present in theseiron-complexes reinforces the practical application of the method.

Since the AHS, referred to as the primary reference standard foriron-saccharidic complexes, can begin to degrade shortly after synthesisor manufacture it can also be useful to establish a secondary referencestandard. In practical terms, the secondary reference standard is basedon the relative occurrence of iron aggregate derived from the activehematinic species compared to the total amount of iron aggregate capableof being released by the active hematinic species under set conditionsover time. The measurement of iron aggregate is justified forestablishing such benchmarks of active hematinic species integrity andstability because detectable iron aggregate levels are formed at theexpense of the primary reference standard. The[AHSAP_(TAM)]/[AHSAP_(TOTAL)] ratio plotted versus time afteriron-saccharidic complex manufacturing or commercial release, providesan index of product storage stability. The time required to reach anarbitrary or performance-related ratio, e.g., 0.5, based on the HPLCsignal quotient of [AHSAP_(TAM)]/[AHSAP_(TOTAL)] can be especiallyuseful, although any ratio can be selected as a guardrail to ensureactive hematinic species product quality. Whatever ratio is selected toserve as a minimum acceptable standard to monitor clinical efficacy,utility and safety based on historical use will set the compositionalstandard for the secondary reference standard. Such a primary referencestandard or a secondary reference standard, can be prepared as practicalanalytical standards for use in monitoring inter- and intra-laboratoryor manufacturing performance and as a product quality index and productstandardization tool.

The separated AHS-containing composition, Fraction 1, comprising theprimary reference standard can be dried for extended storage andreconstituted for parenteral use and additional study.

Dried and/or reconstituted AHS can be stored for purposes of advancedanalytical characterization, for example, in order to establish moredefinitive chemical criteria, as well as archiving AHS samples forfuture reference. Storage of the separated and lyophilized AHS isimportant because iron-saccharidic complexes are subject todestabilization and decomposition following their synthesis,particularly when such complexes remain in a diluent or liquid,particularly aqueous, carrier. In contrast, the dried AHS can be storedfor extended periods of time, preferably in a moisture-free environment,including sealed containers. Furthermore, the dried, stable complex canbe conveniently transported and reconstituted when needed at the pointof use, thereby further extending its stability until just prior to use.For example, the dried AHS can be sealed in moisture proof containerssuch as metal foil pouches or glass containers, and stored at ambienttemperature (about 20° C. to about 25° C.) or below for extended periodsof time. For example, the dried complex can be stored from shortly aftermanufacture, such as from about one week thereafter, as well as after amoderately short storage period of about 6 months to for as long asabout five years or more after manufacture; extended storage can be fromabout 1 year to about five years; or from about 1 year to about 3 years.During such post-manufacture storage, the AHS can be monitored forstability by comparison of the analytical properties, e.g., thechromatographic signature obtained using HPLC in combination with LLSand RI, of a reconstituted sample to a primary reference standard.

The isolated AHS (Fraction 1), as initially made or at any particulartime thereafter, can be freeze dried (lyophilized) and reconstituted forease of storage and transportation as well as for additional study. As aprerequisite to lyophilization, the iron-delivery vehicle or AHS presentas an iron-saccharidic complex is preferably separated from itscoexisting hydrophilic and other excipients as described previously.Such excipients include excess synthesis reactants, reaction byproducts,waste glucans, polyglucans, saccharidic lactones, degradation byproductsand other substances. In a preferred embodiment, the AHS is separated,in the manner of the primary reference standard species, comprisingFraction 1. By virtue of separating the AHS from coexisting hydrophilicsubstances, there is an increase in the A_(w) value of the fraction orcomposition in which it is present; in other words, the A_(w) valueapproaches 1.0 in the AHS containing fraction.

Freeze drying technology is well known in the food processing industryand has also been employed in the drying of pharmaceuticals. Thetechnology is typically applied in order to dry compositions that arewater-wet, although it is feasible to dry materials or solutes that aredispersed or dissolved in other diluents or solvents, alone or inadmixture, for example, with water, and that are susceptible to freezedrying. Generally, the composition is frozen to a temperaturesignificantly below 0° C. and subjected to a low absolute pressure, inother words, a high vacuum. Heat is carefully introduced in order tocause the ice to sublime. The process has been used to protect heatsensitive materials from thermal damage as well as to prevent shrinkageof porous materials during drying, so that they can be quickly and fullyrehydrated. The present invention provides a method for the separationand lyophilization or freeze drying concentration of active hematinicspecies manufactured for use as parenteral iron delivery vehicles.

During freeze drying, a changing state of unbalance exists between icein the frozen composition, referred to as product ice, and systempressure and temperature conditions. The migration of water vapor fromthe product ice interface occurs only if this state of unbalance existsand the product ice is at a higher energy level than the rest of thesystem. Freeze drying equipment is designed to present a set ofcontrolled conditions effecting and maintaining the preferredtemperature and pressure differences for a given product, therebycausing the product to be dried in the least amount of time.

The limit of unbalance is determined by the maximum amount of heat whichcan be applied to the product without causing a change from solid toliquid state (referred to as melt-back). This may occur even though thechamber pressure is low since the product dries from the surface closestto the area of lowest pressure; this surface is called the iceinterface. The arrangement of the drying, solid composition or particlesabove this interface offers resistance to the vapors released frombelow, thereby raising the product pressure and temperature. To avoidmelt-back, heat energy that is applied to the product closelyapproximates, and preferably does not exceed, the rate at which watervapor leaves the product. Another factor affecting the process is therate at which heat energy applied to the product ice (and carried awayby the migrating vapors) is removed by the condenser refrigerationsystem. By maintaining a low condenser temperature, water vapor istrapped as ice particles and effectively removed from the system,thereby reducing and simplifying the vacuum pumping requirement. Air andother noncondensible molecules within the chamber, as well as mechanicalrestrictions located between the product ice and the condenser, offeradditional resistance to the movement of vapors migrating towards thecondenser.

Four conditions are generally considered essential for freeze drying.These process conditions are as follows: (1) the product is solidlyfrozen below its eutectic point or glass transition temperature; (2) acondensing surface capable of reaching temperatures approximately 20° C.colder than the ice interface temperature is provided, typically lessthan about −40° C.; (3) the vacuum system is capable of evacuation to anabsolute pressure of from about 5 to about 65 microns of Hg (about 0.5to about 10 Pa; preferably from about 1 to about 8 Pa); and, (4) asource of heat input to the product, controlled at from about −60° C. toabout +65° C.; preferably from about −40° C. to about +65° C.; morepreferably from about −30° C. to about +55° C.; most preferably fromabout −25° C. to about +25° C.; typically, a temperature of about +20°C. is employed to provide the heat required to drive water from thesolid to the vapor state (i.e., the heat of sublimation). The physicalarrangement of equipment designed to satisfy these four conditionsvaries widely, and includes individual flask freeze drying apparatus andbatch process freeze drying apparatus. Freeze drying processes aretypically carried out in chambers on a batch basis when exacting controlof the process is required, such as in the chemical and pharmaceuticalindustry. This allows an operator to more precisely control what occursto the product being sublimed. Suitable equipment is described, forexample, in U.S. Pat. No. 6,122,836 (assigned to the Virtis Division ofS.P. Industries, Inc., N.Y.) and references cited therein, as well asZapsalis and Beck, Food Chemistry and Nutritional Biochemistry, 1985,Chapter 1, pp. 23-26 (all of which are incorporated herein by referenceto the extent permitted). Other suitable commercial equipment andprocess conditions are described in detail in the section entitled“Freeze Drying”, van Nostrand's Scientific Encyclopedia, Eighth Edition,pages 1338-1342, 1995 (incorporated herein by reference to the extentpermitted).

The effectiveness of freeze drying processes is partially dictated bythe triple point curve for water where solid water in the form of iceundergoes a direct transformation into the vapor phase at temperaturesof less than 0° C., and pressures of less than 4.58 Torr (610.5Pascals). Efficient freeze drying is conducted under a pressure (vacuum)of from about 10 microns to about 200 microns Hg; preferably from about40 to 100 microns; more preferably from about 40 to about 80 microns;typically a pressure of about 60 microns is used. The removal of watermolecules existing as (a) ice within a hydrated physical matrix or (b)ice that develops from freezing simple aqueous solutions, ideally givesa dry residual physical matrix free of water or a residue of somedesired water-free solute. However, where hydrophilic solutes, colloids,suspensions or dispersions exist within an ice system, such assaccharidic excipients, they can concentrate within the ice structure asthe ice is subjected to the freeze drying process and the volume ofsolvent or diluent water is reduced. Since such materials become moreconcentrated as the freeze drying progresses, this increasinglydepresses the freezing point of the frozen aqueous system. As thiscondition proceeds, the colligative properties of solute interactionwith water can also rise above the eutectic point, contributing to orcausing a melt-back phenomenon. This is contrary to the preferred freezedrying process of the present invention in which the ice accompanyingthe desired solute, the AHS, is maintained in a frozen state,substantially unimpaired by hydrophilic solute species that may includedifficult-to-remove water, until substantially all water associated withthe AHS has been removed by sublimation.

The preferred freeze drying of the AHS is accomplished, in significantpart, as a result of the high A_(w) of the fraction (Fraction 1)comprising substantially all of the iron-saccharidic complex originallypresent in the sample, which facilitates its rapid shell-freezing onto aplate, the walls of a container or some other three-dimensionalscaffolding that ensures a high surface to volume ratio for the frozenfraction. The more efficiently that shell-freezing occurs, the betterthe quality of the lyophilized product. Freezing is typically carriedout at temperatures of from about −160° C. to about −10° C.; preferablyfrom about −80° C. to about −20° C.; for example about −60° C. When theAHS is present in a frozen composition where the water displays an A_(w)value approaching 1.0, pressures below about 4.58 Torr (610.5 Pa) resultin increased water vapor pressure and temperature conditions asdescribed can result in an increased water vapor pressure and efficientsublimation. Water is removed from ice by maintaining the pressuresurrounding the frozen. AHS below the vapor pressure on the surface ofremaining ice, removing the water vapor with a vacuum pump andcondensing it on refrigerated surfaces held at temperatures of fromabout −120° C. to about −25° C.; preferably from about −80° C. to about−50° C.; typically −60° C. In particular, the high A_(w) of thepreviously separated AHS facilitates the migration rate of thesublimation front throughout the frozen product. In the absence ofremoving the excipients, particularly hydrophilic excipients, from theAHS or iron-saccharidic complex, the AHS is subject to melt-back duringthe freeze drying process. In other words, the presence of hydrophilicsubstances results in water being sufficiently bound or retained by theAHS composition in which such hydrophilic substances are present. Ifhigher temperatures are employed to increase the vapor pressure in aneffort to remove such bound water, this also can have the undesirableeffect of causing the ice phase to melt, thereby impairing freezedrying. Consequently, it is preferred that all or substantially all ofthe hydrophilic excipients be removed or separated from the AHS prior tofreeze drying: preferably greater than about 95% of those originallypresent are removed; more preferably greater than about 98%; still morepreferably greater than about 99%; most preferably greater than about99.9% are removed; for example, the AHS is separated from hydrophilicexcipients prior to freeze drying to the extent that such excipients arepresent in trace amounts.

For purposes of the present invention, the dried AHS residue comprisesthe pharmacologically useful iron-saccharidic complex. Thus, theseparated and dried AHS is suitable for further analytical study or,optionally, reconstitution, in order to meet other investigativeanalytical or pharmacological uses. Typically, the methods of thepresent invention are suitable for drying AHS, from which excipientshave been substantially removed, such that from about 85% to at leastabout 99%; preferably from about 90% to at least about 97%; mostpreferably from about 92% to at least about 95% of the water has beenremoved. It should be appreciated that a small percentage of the wateroriginally present in the separated AHS may be associated, or stronglybound, to the AHS and attempts to remove such bound water may pose adanger of unnecessarily degrading the AHS. A sample of post-lyophilizedand reconstituted AHS subjected to LLS and RI-based HPLC analysis, isillustrated in FIG. 10. The figure shows a chromatographic signaturesubstantially identical to that in FIG. 1, which serves as the primaryreference standard. Moreover, the analytes depicted in FIGS. 1 and 10are essentially identical to the AHS seen in FIG. 3 where excipientswere allowed to remain.

A hematinic material with an HPLC profile that is different from theprimary reference standard such as in FIG. 1, or in FIG. 10, is one thatalso shows evidence of the AHSAP in the lyophilized product having anunusual morphology. When the AHSAP is observed to be present as part ofthe AHS in the course of HPLC studies, the microscopic appearance of thelyophilized AHS at 100-fold magnification and higher, can be visuallydescribed as a corduroy-type structure. It displays red-brown parallelbands or wales of ferric iron uniformly interspaced with transparentbands of thin carbohydrate plates. The red-brown parallel bands offerric iron have a distinct columnar shape, parallel to each other, thatare at least twice the diameter of the thickness displayed by the longplanar transparent carbohydrate plates that are repeatedly interspacedbetween them. This observed microscopic form has structural analyticalsignificance that corresponds to HPLC light scattering data whenchromatographic profiles appear as those seen in FIGS. 4-9. When adesirable freshly prepared AHS corresponding to the primary referencestandard of iron-saccharidic complexes is present, the morphology of thelyophilized product is characteristically different in that there is anabsence of columnar structure.

Preservation of the lyophilized product can be maintained in a vacuum orunder any inert gas, including, for example, nitrogen, argon and helium(as well as any gas that is not reactive with the lyophilized product)before it is reconstituted for analysis or use. Also, since thelyophilization process alone does not compromise the structure ofiron-saccharidic complexes, use of the process has value for maintainingthese hematinic agents at various time intervals so as to document thehematinic species present at a given point in time when lyophilizationwas implemented. This provides a method for archival storage anddocumenting of product manufacture and quality. In other cases,lyophilization can be specifically used to stabilize and store primaryand/or secondary reference standards of these hematinic compositions.Furthermore, suitably prepared and maintained lyophilized AHS can besafely stored until needed with little risk of significant degradationof the product. Furthermore, the product in such a form can beconveniently shipped to geographically remote locations and convenientlystored until needed, at which time reconstituting the hematinic forparenteral use is readily accomplished. For example, the lyophilizedproduct prepared according to the present invention can be stored insealed glass or appropriately protected metal containers, preferablytopped with a substantially moisture free inert gas. Alternatively, suchproduct can be sealed in a metal foil pouch in a quantity suitable forreconstituting as a single parenteral dose, etc. The iron-saccharidiccomplexes referred to are prepared in order to produce parenteralhematinic complexes for the delivery of iron to humans or animals inneed thereof. These iron complexes generally occur in a form such thatiron can be parenterally and benignly administered to augmenthematopoietic mechanisms required for the management of numerousclinical conditions in mammals, particularly in human beings in needthereof.

Parenteral administration of a substance, e.g., a drug or the AHS of thepresent invention, refers to introduction into the body by some meansother than through the gastrointestinal tract. In particular, itincludes, intravenous, subcutaneous, intramuscular or intramedullaryinjection or short, e.g., about 5 minutes, or prolonged infusion, e.g.,about 30 minutes or longer. Parenteral routes of administration canprovide benefits over oral delivery in particular situations. Forexample, parenteral administration of a drug typically results inattainment of a therapeutically effective blood serum concentration ofthe drug in a shorter time than is achievable by oral administration.This is especially true of intravenous injection, whereby the drug isplaced directly in the bloodstream. Parenteral administration alsoresults in more predictable blood serum concentrations of the drug,because losses in the gastrointestinal tract due to metabolism, bindingto food and other causes are eliminated. For similar reasons, parenteraladministration often permits dose reduction. Parenteral administrationis generally the preferred method of drug delivery in emergencysituations, and is also useful in treating subjects who areuncooperative, unconscious, or otherwise unable or unwilling to acceptoral medication. With regard to hematinics, parenteral administration ofthe AHS is particularly useful for patients undergoing dialysistreatment since it can be administered concurrently.

As described above, the separated AHS can be lyophilized and stored as afreeze-dried composition. Thereafter an injectable solution can beprepared by reconstitution of the composition. Furthermore an article ofmanufacture can be produced comprising a sealed container such as avial, ampoule or pouch having contained therewithin a unit dosage amountof the composition in a sterile condition. Such an article can be usedfor treating or preventing an iron deficiency disorder in a subject, themethod comprising (a) reconstituting a unit dosage amount of thecomposition in a physiologically acceptable volume of a parenterallyacceptable solvent liquid to form an injectable solution, and (b)injecting the solution parenterally into the subject. At the time thatthe AHS is reconstituted for parenteral use, additional agents orexcipients can be added in controlled amounts in order to provide asuitable parenteral solution. Such agents include, for example,buffering agents, pH modifiers, preservatives, tonicity adjustingagents, etc. Alternatively, one or more of such agents can be includedin an appropriate amount in dry or powder form with the lyophilized AHSsuch that when the AHS is reconstituted for parenteral use, theresulting parenteral composition includes the necessary materials toform a suitable parenteral composition for immediate use. Alternatively,only the buffering agent is present and other agents are added to theextent required.

One or more active hematinic species selected from those disclosedhereinabove are present in a reconstitutable powder composition of theinvention in a total amount of about 30% to about 95%, alternativelyabout 40% to about 90%, or about 50% to about 85%, by weight of thecomposition.

When used, the buffering agent is present in an amount of about 5% toabout 60%, preferably about 10% to about 60%, and more preferably about20% to about 50%, by weight of the composition, and is typically thepredominant excipient ingredient. In one embodiment of the invention,the reconstitutable powder composition consists essentially of the AHSand the buffering agent.

The buffering agent is selected to provide a pH of the composition, uponreconstitution in a physiologically acceptable volume of a parenterallyacceptable carrier or solvent liquid, that (a) is parenterallyacceptable, (b) is consistent with the AHS being in solution orsufficiently dispersed so as not to cause an unacceptable adversereaction, in the carrier or solvent liquid, and (c) provides a mediumwherein the AHS exhibits acceptable chemical stability for at leastabout one hour following reconstitution so as to facilitate parenteraladministration. Suitable buffering agents can illustratively be selectedfrom sodium and potassium phosphates, sodium and potassium citrates,mono-, di- and triethanolamines,2-amino-2-(hydroxymethyl)-1,3-propanediol (tromethamine), etc. andmixtures thereof. Preferred buffering agents are dibasic sodium andpotassium phosphates and tromethamine. An especially preferred bufferingagent is dibasic sodium phosphate, for example dibasic sodium phosphateanhydrous, heptahydrate, dodecahydrate, etc.

In one embodiment, the pH of the composition upon reconstitution isabout 7 to about 9, preferably about 7.5 to about 8.5, for example about8. If desired, pH can be adjusted by including in the composition, inaddition to the buffering agent, a small amount of an acid, for examplephosphoric acid, and/or a base, for example sodium hydroxide.

Excipients other than the buffering agent, if present, constitute notmore than about 10%, preferably not more than about 5%, by weight of thecomposition prior to reconstitution. For purposes of this discussion,the term excipient does not include water. In one embodiment of theinvention, no excipients other than the buffering agent aresubstantially present.

Optionally, one or more preservatives can be included in the compositionat up to about 0.5% by weight. Suitable illustrative preservativesinclude methylparaben, propylparaben, phenol and benzyl alcohol.

An injectable solution composition prepared by reconstituting a powdercomposition as herein provided in a parenterally acceptable liquidcarrier or solvent, preferably an aqueous solvent, is a furtherembodiment of the present invention. Any known parenterally acceptableliquid carrier or solvent can be used to reconstitute a powdercomposition of the invention. Water for injection can be suitable, butwill generally provide a hypotonic solution. Accordingly, it isgenerally preferred to use an aqueous liquid containing a solute such assodium chloride and/or possibly dextrose. Illustratively, 0.9% sodiumchloride injection USP, sterile 0.9% sodium chloride injection USP, 5%dextrose injection USP, and 5% dextrose and 0.45% sodium chlorideinjection USP are suitable.

A suitable volume of the liquid carrier or solvent for reconstitutiondepends on the age and body weight of the subject, the solubility anddosage amount of the AHS and other factors, such as whether theparenteral composition is to be administered by injection, IV push orIV.

In this process, AHS and dibasic sodium phosphate heptahydrate asbuffering agent are dissolved in water to form an aqueous solution orcomposition. Preferably water for injection is used as the solvent. AHSand the buffering agent are present in the solution at concentrationsrelative to each other consistent with the desired relativeconcentrations of these ingredients in the final composition. Absoluteconcentrations of these ingredients are not critical; however, in theinterest of process efficiency it is generally preferred that theconcentration of AHS be as high as can be conveniently prepared withoutrisking exceeding the limit of solubility to the extent of forming anunsuitable aggregate. Other parenteral formulation ingredients or agentsas described above can be added in this step if desired. Order ofaddition is not critical

An article of manufacture comprising a sealed vial, preferably a glassvial, having enclosed therewithin a powder composition as hereinprovided in a unit dosage amount and in a sterile condition, is afurther embodiment of the present invention. In a particular embodiment,such an article of manufacture is provided, prepared by a process asdescribed above. The vial preferably has a capacity sufficient to enablereconstitution of the composition in situ. Generally a capacity of about1 ml to about 10 ml, preferably about 2 ml to about 5 ml, will be foundconvenient. The term “vial” herein is used to denote any smallcontainer, having a closure, that is suitable for packaging a unitdosage amount of a reconstitutable powder, preferably in a sterilecondition. It will be understood that equivalent forms of packaging,such as an ampoule, a disposable syringe and a syringe cartridge, areencompassed by this embodiment of the invention.

The present invention is further directed to a therapeutic method oftreating a condition or disorder where treatment with a hematinic isindicated, the method comprising parenteral administration of areconstituted composition of the invention to a subject in need thereof.The dosage regimen to prevent, give relief from, or ameliorate thecondition or disorder preferably corresponds to any suitable interval inaccordance with a variety of known factors. These include the type, age,weight, sex, diet and medical condition of the subject and the natureand severity of the disorder. Thus, the dosage regimen actually employedcan be varied.

A typical preparation comprising an AHS prepared according to theprocess of the present invention and provided in a suitable container,e.g., an ampoule, vial or pouch, generally contains sufficient AHS so asto provide, upon reconstitution, about 5 to 100, e.g., about 7 to about50, typically about 10 to about 40 mg iron per mL.

A parenteral AHS in the form of sodium ferric gluconate can be producedin a composition equivalent to that of a presently available commercialproduct, for example, in the presence of sucrose. Consequently, thecomposition can be administered in a dosage form and based on anadministration schedule equivalent to that currently recommended. Thedosage is typically expressed in terms of the mg content of elementaliron. For example, the recommended dosage for repletion of irondeficiency in hemodialysis patients is equivalent to 125 mg of iron fora single administration. The product, when provided in the form of a 5mL ampoule for intravenous injection containing 62.5 mg (12.5 mg/mL) ofelemental iron and also containing approximately 20% sucrose w/v (195mg/mL) in water at a pH of 7.7-9.7, can be administered as a 10 mL dose;equivalent to 125 mg of elemental iron. For slow IV administration(undiluted), 125 mg can be introduced over 10 minutes; for IV infusion(diluted in 0.9% NaCl), 125 mg in 100 mL over 60 minutes. A physiciantrained in the art can determine the appropriate total dosage needed bya patient based on the medical and physical condition of the patient andthe iron improvement required. For example, in order to achieve afavorable hemoglobin or hematocrit response, the current recommendationfor the commercial hematinic of the above type is a minimum cumulativedose of 1.0 gram of elemental iron, administered over eight sessions at,e.g., eight sequential dialysis treatment sessions.

Dosage and administration of a parenteral product based on anothercurrently available commercial product in the form of sodium ferrichydroxide in sucrose is also described in the art. Dosage of this formis also typically expressed in terms of elemental iron content.Typically each 5 mL vial of the composition contains 100 mg of elementaliron based on 20 mg/mL. Repletion treatment of iron deficiency inhemodialysis patients is typically 5 mL comprising 100 mg of elementaliron delivered intravenously concurrent with dialysis. Patientstypically require a total of 1 gram (1,000 mg) of elemental ironadministered in conjunction with 10 sequential dialysis sessions for anappropriate hemoglobin or hematocrit response. Maintenance ofappropriate levels of hemoglobin, hematocrit and other laboratorycriteria may be determined by a skilled physician, as appropriate.

The term “about” when used as a modifier for, or in conjunction with, avariable is intended to convey that the numbers and ranges disclosedherein are flexible and that practice of the present invention by thoseskilled in the art using temperatures, concentrations, amounts,contents, carbon numbers, properties such as molecular weight,viscosity, solubility, etc., that are outside of the range or differentfrom a single value will achieve the desired result, namely preparationof a hematinic iron-saccharidic complex suitable for freeze drying, thehighly purified hematinic iron-saccharidic complex produced thereby andmethods for its use. Furthermore, where a range of values is expressed,it is to be understood, unless otherwise expressed, that the presentinvention contemplates the use of the other ranges that are subsumedwithin the broadest range.

EXAMPLES

For purposes of the present invention, reference to water content of anundried substance or composition, in other words, prior to being dried,is given as a percentage of the total weight of the undried substance orcomposition. Water content of a dried substance or composition is givenas a percentage of the total weight of dry matter only, excluding allmoisture.

Following is the procedure for low pressure gel permeationchromatography used in preparing the samples for which test resultsappear in FIGS. 1, 2 and 10, including preparation of purified,substantially excipient-free AHS. The specific application of lowpressure gel permeation chromatography (GPC) for AHS separation employscrosslinked polyglucans or dextrans displaying molecular weightexclusion characteristics greater than about 5,000 and preferablygreater than about 1,500 Daltons. The stationary GPC phase is “SephadexG-10” (Amersham-Pharmacia Biotech, Piscataway, N.J.). A solventreservoir supplies a mobile phase of HPLC grade water by gravity ormetered flow to a GPC column containing the stationary dextran phase.The column is constructed of glass having a 2.0 cm diameter and a lengthof 25 cm. The stationary phase is prepared according to manufacturerrecommendations including hydration of the dextran and vacuum degassingbefore use. A 400 microliter sample volume of the hematinic solution,including the iron-saccharidic complex, for example, as released by itsmanufacturer in sealed glass ampoules, is fed to the top of the GPCcolumn and allowed to permeate into the stationary phase. Once thehighly colored hematinic solution has penetrated into the stationaryphase, HPLC grade water is supplied manually or by pump at 1-4 mL perminute so as to ensure its elution as a well-defined color band throughthe column. When the characteristically colored AHS has eluted from thecolumn as determined by minimal spectrophotometric absorption at 430 nmthis marks the end of elution for Fraction 1. A more refined analyticalmethod for finding a separation point for Fraction 1 and the beginningof the subsequent fraction identified herein as Fraction 2 uses theanthrone reaction (Dreywood, 1946 cited previously). The eluate boundarybetween the two Fractions can be defined because the lowestconcentration of furfural-producing carbohydrates in the overall eluateflow of the partitioning process occurs between the AHS and itshydroscopic excipients. In practice, 100 microliter samples of theeluate flow are sampled, reacted with anthrone reagent and the resulting620 nm absorbance is recorded. The red-brown colored Fraction 1 containsthe AHS substantially free of hydrophilic and highly hydroscopicexcipients that formerly coexisted with the AHS as released. Remainingvolume eluted from the column is regarded as Fraction 2.

The AHS obtained, for example, from Fraction 1 as described above, orfrom samples taken directly from glass ampoules of hematiniccompositions distributed for use in clinical applications or samplesprepared from concentrated volumes of Fraction 1, includingreconstituted compositions based on the use of freeze drying, can befurther analyzed with the use of HPLC-based refractive index (RI) andlaser light scattering (LLS) analysis. Specifically, the method uses aWaters 590 pump (Waters Corporation, Milford Mass.) to supply an aqueousmobile phase to a 7.8 mm diameter by 30 cm long GMPW_(XL) column (TosohBiosep, Montgomeryville, Pa.). The internal column support material iscomprised of polymethylmethacrylate backbone eliminate polymer beadshaving a 13 micron diameter particle size with a various pore sizes inthe range of from less than about 100 Angstroms to about 2000 angstroms.The column eluant stream is monitored by a Wyatt miniDawn multi-anglelight scattering detector in combination with an Optilab DSPinterferometric refractometer (both from Wyatt Technology, Inc., SantaBarbara, Calif.). The column heater and refractometer operatingtemperatures were held at 35° C. The aqueous mobile phase included 200parts per million sodium azide, pH was adjusted to 6.0 and was subjectedto 0.02 micron vacuum filtration and an ebullient helium sparge beforebeing used. The mobile aqueous phase was supplied to the system at aflow rate of 1.0 mL per minute with a pressure of 150 pounds per squareinch. Preparation of a sample for testing requires 0.02 micronfiltration through a membrane filter (for example, “Anotop” filters,Whatman, Maidstone, England). As hematinic compositions,iron-saccharidic complexes or components thereof age, membrane filtersup to 0.45 microns may be required in order to remove largerparticulates without clogging. If particulates are not eliminated fromanalytical samples before injection into the HPLC system, HPLCanalytical performance will be severely corrupted. Samples are dilutedas desired up to 2.5% weight by weight and a 80 to 200 microliter samplevolume is injected into the HPLC system for analysis. For multiplesample analyses, automation is facilitated by use of “Water'sautosampler”, model 717 (Milford, Mass.).

The combination of RI and LLS detection with HPLC establishes anabsolute macromolecular weight for analytes that produce achromatographic peak as well as a root mean square (rms) radius valuealso referred to as a radius of gyration (R_(g)). The rms value coupledwith absolute weight determination provides insight into the shapes oflight scattering species, such as rods, coils, spheres or discs. Theformula weight of the AHS and shape of specific iron-saccharidiccomplexes can be used for various monitoring purposes.

The freeze drying process used for the examples of the present inventionis as follows:

Fraction 1, identified above, serves as the starting material for freezedrying. Using the method described below, volumes as small as 10 mL oras large as 100 mL and comprising the AHS can be readily freeze driedprovided that the sample is substantially free of saccharidic substancesthat tend to decrease the entropy of water and its vapor pressure. Thesevolumes can be contained in any glass container that will withstand thephysical stress of shell freezing, which method is used to expedite theoverall dehydration and concentration of the AHS. The preferred ratio ofliquid volume to the container volume for shell freezing is from about 1to about 5, but other ratios are feasible. After liquid containing thesubstantially excipient-free AHS is introduced into the container, thecontainer is rotated at about 50 revolutions per minute in a cryogenicbath. The bath can be made by mixing dry ice and acetone or,alternatively, liquid nitrogen can be used, provided that a temperatureof at least about −50° C. or lower is maintained. The immersion androtation of the container freezes the AHS-containing aqueous volume ontothe walls of the container. This increases the surface to volume ratioof the AHS-containing aqueous volume so as to expedite watersublimation. Other process and equipment variations of this procedurecan be used to obtain the same or similar results.

One or more containers of shell-frozen compositions comprising water andAHS are situated on a shelf within a freeze dryer. An instrument such asa “Virtis Unitop 600L” linked to a “Freezemobile 12 ES” (Gardiner, N.Y.)can be used for this purpose. A vacuum of 60 microns of Hg (7.5 Pa) wasmaintained in the system and a condenser temperature of at least about−60° C. or colder was maintained. A preferred freeze drying cycle was asfollows: initial shelf holding temperature of −50° C. for 2 hours;temperature ramped up to 25° C. over a 12 hours; sample soak at 25° C.for an additional 24 hours. Preferably the dried product should bestored under desiccating storage conditions, for example under a dry,inert gas such as argon or nitrogen. The dried AHS can be reconstitutedwith a desired aqueous volume whereupon it readily goes into a solutionand can be readily filtered through a 0.02 micron membrane, as describedabove.

HPLC analysis using RI and LLS detection is demonstrated in thefollowing 10 examples using iron-saccharidic complexes. The results foreach of samples 1 through 10 corresponds to FIGS. 1 through 10. Thehematinic samples 1-4 and 6-10 are the iron-saccharidic complexidentified as sodium ferric gluconate complex in sucrose (SFGCS), soldunder the brand name Ferrlecite® (manufactured by Rhone-Poulenc Rorer,Dagenham, Essex, England). Sample 5, and its corresponding FIG. 5, isferric hydroxide sucrose complex (FHSC), sold under the brand nameVenofer® (manufactured by Luitpold Pharmaceuticals, Shirley, N.Y.).Samples used for HPLC analysis were taken from newly opened glassampoules stored at room temperature conditions. Additionally, samples 6,7, 8, and 9 were analyzed after 6, 12, 22 and 25 months followingmanufacturing release of the product. These time periods are referred toherein as “time after release”, TAM and correspond to TAM_(#1),TAM_(#2), TAM_(#3) and TAM_(#4) in the examples. Samples were preparedby a 1 to 20 dilution and 200 microliters sample volumes of thesedilutions were analyzed by the HPLC method specified above.

Example 1 test results are shown in FIG. 1. The results are based on theuse of HPLC with RI and LLS detection for evaluating an AHS isolated inthe Fraction 1 eluate obtained using preparative low pressure GPC and asample of iron-saccharidic complex as obtained from its glassdistribution ampoule. The single well defined chromatographic profilefor the LLS signal and the RI signal coincide for AHS elution but noother excipients appear in the purified material.

Example 2 results are shown in FIG. 2. The results are based on the useof HPLC with RI and LLS detection for evaluating the AHS isolated in theFraction 2 eluate obtained from using preparative low pressure GPC andthe iron-saccharidic complex of Example 1. It is clear from these tworesults that the excipients and AHS are separated or isolated indistinct fractions. 15 microliters of AHS from Fraction 1, Example 1 wasadded to the Fraction 2 eluant as an internal standard in order toidentify where its elution position would appear relative to that of theexcipients.

Example 3 results are shown in FIG. 3. The results are based on the useof HPLC with RI and LLS detection, applied to the same hematinic sampleas in Example 1, to substantially separate Fraction 1 with itscharacteristic AHS, from excipients usually observed in Fraction 2. TheHPLC method can discern the various iron-saccharidic complexconstituents of a hematinic composition on a single chromatographicprofile. While HPLC is particularly suited to rapid analytical testing,low pressure chromatography is particularly suited as a preparativemethod for the preferred AHS. The LLS signal for the AHS corresponds tothe observed RI signal.

Example 4 results are shown in FIG. 4. The results are based on the useof the preferred HPLC method for detecting structural deviations fromthe original AHS. In its undegraded form, the AHS serves as a qualitybenchmark also denoted in these Figures as a “primary referencestandard”. HPLC analysis using. RI and LLS was carried out on ahematinic composition obtained directly from its delivery ampoule andcomprising an iron-saccharidic complex. The figure shows inconsistenciesin the expected, or ideal, AHS peak. Note the appearance of a new,observable chromatographic secondary peak adjoining that of the AHSprimary reference standard. This feature is indicative of iron aggregatespecies as a consequence of AHS degradation. The figure identifies thesecond peak as an active hematinic species aggregate peak (AHSAP) usingLLS detection. It is particularly noteworthy that this peak is notobserved using RI detection alone.

Example 5 results are shown in FIG. 5. The results are based on the useof HPLC equipped with RI and LLS detectors. This figure also showsstructural changes in the AHS, or primary reference standard peak, ofhematinic comprising FHSC. The sample of this product included amanufacturing date of December 1999 on the ampoule and an expirationdate of December 2002. In this example, the AHSAP appears as a shoulderon the AHS peak, suggesting a different degree of change compared withthe sample studied in Example 4. While LLS and RI chromatographicprofiles generally overlap, only the LLS signal detects evidence of ironaggregates in the parenteral hematinic. As in each previous example, thesample for this study was obtained directly from a sealed glass ampouleused for clinical distribution.

Examples 6-9. Readily detectable departures in the HPLC based RI and LLSelution profiles from the expected chromatographic profile for an AHSreflect either a departure from preferred manufacturing conditions ordegradation of AHS due to aging and destabilization while still sealedin glass delivery ampoules. The destabilization reflects itself in theaggregation of iron normally embodied as a constituent of the desirableAHS structure. Although HPLC with RI detection fails to detect thechanges in the iron-saccharidic complexes, LLS detection clearlyprovides evidence of such product destabilization. The advantages of theinvention as a method for monitoring the state of a hematinic productcomprising iron-saccharidic complex is illustrated where HPLC is coupledwith RI and LLS detectors in order to detect evidence of degradation ofthe AHS as indicated by iron aggregate formation. Iron aggregateformation is observed as the HPLC chromatographic peak denoted as AHSAP.Individual samples of iron-saccharidic complexes, specifically SFGCS,manufactured over the course of several months were stored at roomtemperature in the absence of light and without any excursions known tostress the stability of the products while sealed in their glassampoules. The samples were aged at room temperature in the dark andafter 6, 12, 22 and 25 months following their manufacture, therespective ampoules were opened and the contents analyzed by HPLC withRI and LLS, as described. None of the stored product samples had reachedits stated expiration date stated on the packaging material. The samplewith the shortest TAM value of 6.0 months was designated as TAM_(#1) andthat with the longest storage of 25 months TAM_(#4). The results of HPLCstudies applied to this range of successively aged hematinic examplesare shown in FIGS. 6-9. The key area of analytical interest in thechromatographic profiles presented in FIGS. 6-9 is the region where theAHS signature appears, thus only that specific elution range pertinentto the RI and LLS analytical profile is shown.

Taken as a group in sequence from TAM#1 (FIG. 6) through TAM#4 (FIG. 9),it is apparent that the RI signal from these samples show little effectof sample aging by way of AHS decomposition and iron aggregation. On theother hand, evidence of AHS destabilization with iron aggregation isprominently seen by the AHSAP shoulder or a secondary peak, which wasdetected by using LLS in all four samples.

By way of these examples, it is evident that the preferred HPLC based RIand LLS method provides an ability to verify the presence of an AHS orits coexistence with normally occurring excipients with which it isreleased for parenteral use. Beyond this, the method affords asignificant ability to monitor, as well as to investigate and develop,hematinics based on iron-saccharidic complexes as a group. It can beseen that this class of hematinics is susceptible to destabilizationresulting in iron aggregates that are commingled with the preferred ornormal AHS. Unless HPLC is used with at least LLS detection as well asRI detection, the occurrence of iron aggregates can go unnoticed inthese hematinic agents.

As described above, when carrying out the preferred method of thisinvention, samples are routinely filtered through a 0.02 micron Anotopbrand membrane filter to avoid operational problems with the samplebefore it is injected into the HPLC. Iron-saccharidic samples that showno evidence of AHSAP occurrence can be readily filtered in preparationfor study but older samples filter with great difficulty or not at alleven using 0.45 micron filters. It has been observed that difficultiesin the preparatory filtration of samples for HPLC study according to thespecified and preferred method correspond with the occurrence of thehighest levels of AHSAP. The measurable and quantitative entrainment ofiron aggregates over a membrane filter surface can provide another,albeit cursory, method for evaluating the unintended degree of hematinicbreakdown. However, application of such a filtration method to ahematinic before parenteral use would be impractical and, furthermore,it would not have any effect on degraded AHS that had not progressed tothe filterable iron aggregate stage. When significant amounts of ironaggregates develop in samples and hematinic analysis is essential,sample filtration is necessary for HPLC instrument performance andmaintenance. Also, it should be noted that if a residue of ironaggregate or other particulate material is entrained on a membranefilter as a result of preparing AHS for HPLC analysis, the quantitativeamount of aggregate on the filter should be considered together withHPLC analysis as complementary indicators of product decay. Where littleor no evidence of filterable material is present in hematinics andparticulates have dimensions of less than 10 nm in diameter, the HPLCmethod more accurately serves as the preferred method for monitoringhematinic quality.

Example 10 results are shown in FIG. 10. This example involved theapplication of the preferred method for HPLC based RI and LLS analysisto an AHS that was reconstituted from a freeze dried state. While AHSisolated from an iron-saccharidic complex has never before beenreported, its ability to be freeze dried and reconstituted withoutdecomposition, degradation or iron aggregate formation was particularlyuncertain. An original 2.5 mL sample volume of iron-saccharidic complexas released, SFGCS, taken from a sealed glass ampoule was separated intoFraction 1 and Fraction 2 according to the low pressure chromatographymethod for preparing substantially excipient free AHS. Fraction 1containing the AHS was freeze dried as described above. One week afterfreeze drying it was reconstituted to its original volume (2.5 mL) withHPLC grade water. A 500 microliter volume of the 2.5 mL reconstitutedsolution was then diluted to 20.0 mL and 200 microliters of this wasinjected for HPLC based RI and LLS analysis as described. The resultingchromatographic profile is shown in FIG. 10. It is noted that both theLLS and RI signals not only overlap, consistent with the chromatographicprofiles observed in FIG. 1, but there is no evidence of any ironaggregate formation or AHSAP as observed in FIGS. 6-9. By way of thisexample, HPLC with RI and LLS detection is also shown to be useful formonitoring the quality of freeze dried AHS.

Example 11 was carried out to compare the response to freeze drying ofuntreated versus treated hematinic. Three samples of an iron-saccharidiccomplex, SFGCS, as manufactured and comprising AHS, sucrose and residualexcipients were subjected to freeze drying. All three samples originatedfrom the same production batch as released in different glass ampoules.The freeze drying method used was the same as that applied to thehematinic in Example 10, but contrary to that example, the AHS wasallowed to remain with its excipients during the course of freezedrying. The experimental objective was to observe whether or not therewere any weight disparities in the final freeze dried product due to thepresence of hydrophilic excipients compared to the same product freezedried in the absence of such hydrophilic excipients. Results aresummarized for the hematinic dried with its excipients in Table A.

TABLE A Sample Water removed Freeze dried No. Sample weight (g) (%)*weight (g) 1 10.414 77.3 2.373 2 10.330 79.9 2.169 3 9.481 78.2 2.070Mean ± sd 10.075 ± 0.421 78.5 ± 1.1 2.204 ± 0.126 *Expressed as apercent of original sample weight

Three additional samples of the same hematinic batch from the samesource as used in Table A were obtained from different unopened ampoulesand subjected to treatment in a low pressure chromatographic column asdescribed above. Fraction 1 of each sample (comprising the AHS, butwithout the associated hydrophilic excipients, including sucrose) wasthen subjected to freeze drying as described above; results aresummarized in Table B:

TABLE B Sample Water removed Freeze dried No. Sample weight (g) (%)*weight (g) 1 9.500 93.50 0.613 2 7.840 93.70 0.498 3 3.200 91.47 0.273Mean ± sd 6.847 ± 2.667 92.89 ± 0.10 0.461 ± 0.140 *Expressed as apercent of original sample weight

The test results clearly show that a significantly higher percentage ofwater is removed from the samples subjected to column separation andidentical conditions of freeze drying.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

1. A method for measuring at least one molecular characteristic of aniron-saccharidic complex present in a composition comprising one or moreexcipients, said complex comprising at least one active hematinicspecies (AHS), said method comprising: (A) using a first portion of saidcomposition and substantially separating said AHS from said one or moreexcipients to obtain purified AHS; determining the differentialrefractive index increment (dn/dc) of said purified AHS, (B) subjectinga second portion of said composition to liquid chromatographic analysis(LCA) having a refractive index (RI) detector and in-line eluate streamdetection using laser light scattering (LLS); and (C) calculating saidat least one molecular characteristic based on said LCA and said dn/dcvalue.
 2. he method of claim 1 wherein said LLS comprises multi-anglelaser light scattering (MALLS), low angle laser light scattering (LALLS)or a mixture of MALLS and LALLS.
 3. The method of claim 1 furthercomprising the use of one or more of the following tests in step (A):electrochemical detection (ECD), photodiode array (PDA) based UV-VISspectrophotometry, infrared (IR) spectroscopy, and liquid chromatographycoupled with mass spectrometry (LC-MS).
 4. The method of claim 3 whereinsaid at least one molecular characteristic is selected from the groupconsisting of absolute molecular weight, molecular weight distribution,size, shape, morphology, and dimensional variations.
 5. The method ofclaim 1 wherein said at least one or more excipients comprises anon-hematinically active component.
 6. The method of claim 5 whereinsaid non-hematinically active component is selected from the groupconsisting of iron-saccharidic complex synthesis reaction by-products,unreacted iron-saccharidic complex synthesis starting materials,iron-saccharidic complex degradation by-product, waste glucans,polyglucans, saccharidic lactones, solvent and diluent.
 7. The method ofclaim 1 wherein said step of substantially separating is selected fromthe group consisting of electrokinetic migration, electrokinetic-basedmembrane separation, capillary electrophoresis, and columnchromatography.
 8. The method of claim 7 wherein separating comprisespassing said iron-saccharidic complex through a chromatographic columnand separating the column eluate into fractions.
 9. The method of claim8 wherein said column is selected from the group consisting of a highpressure liquid chromatography column and a size exclusionchromatography column, each column comprising a stationary phase. 10.The method of claim 9 wherein said stationary phase in said sizeexclusion chromatography column comprises crosslinked dextran.
 11. Themethod of claim 1 wherein in addition to said at least one activehematinic species said composition further comprises an iron aggregatehaving a molecular weight higher than said at least one active hematinicspecies and said LCA further comprises: (1) identifying an elutioncomposition profile of said composition, said elution profile comprisingat least a first eluate comprising said iron aggregate, a second eluatecomprising the at least one active hematinic species of saidiron-saccharidic complex and a third eluate comprising excipients havinga molecular weight lower than said at least one active hematinicspecies; and (2) separating said first and third eluates from saidsecond eluate.
 12. The method of claim 11 wherein said elutioncomposition profile is determined using at least one additional detectorselected from an ultraviolet-visible light transmission detector or avisible light detector for detection at one or more defined wavelengths.13. The method of claim 1 wherein said AHS is selected from the groupconsisting of sodium ferric gluconate complex, ferric hydroxide sucrosecomplex and ferric saccharate complex.
 14. The method of claim 1 whereinsaid at least one molecular characteristic is absolute molecular weight,or molecular weight distribution or both.
 15. The method of claim 1wherein said purified ANS in step (A) is lyophilized.